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Bovine BRCA1 shows classic responses to genotoxic stress but low in vitro transcriptional activation activity

Oncogene volume 22, pages 6032–6044 (2003)Cite this article

Abstract

Human BRCA1 has a genetically demonstrated role in DNA repair, and has been proposed to act as a transcriptional activator in a limited number of specialized settings. To gain insight into biologically conserved functional motifs, we isolated an ortholog of BRCA1 from cattle (Bos taurus). The predicted protein product shows 72.5% sequence identity with the human protein and conservation of amino acids involved in BRCA1 structure and function. Although the bovine C-terminus is truncated by seven amino acids as compared to human, bovine BRCA1 protein exhibited a similar cell cycle-regulated nuclear expression pattern. Expression was characteristically low and diffuse in the nucleus of G1/G0 cells, followed by increasing BRCA1-positive nuclear speckles in late S phase and G2/M phase cells. Bovine BRCA1 was phosphorylated and nuclear speckling was enhanced in response to DNA-damaging agents. Consistent with evidence from studies of human BRCA1, bovine BRCA1 was shown to interact with RNA polymerase II in vivo, an activity that was mapped to the C-terminal domain (CTD) (bBRCA1364–1849). Interestingly, when tested in the GAL4 transcriptional activation assay, full-length bovine and human BRCA1 lacked any ability to act as transcriptional activators and the CTD of bovine BRCA1 had five-fold lower activity when compared to the more acidic human C-terminus. These results provide evidence that phosphorylation and nuclear relocalization are highly conserved features of the BRCA1 response to genotoxic stress. In addition, bovine BRCA1 binds the RNA polymerase II holoenzyme, but this interaction lacks significant ability to correctly orient or recruit RNA polymerase II for transcription in the classic GAL4 transcriptional activation system.

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Acknowledgements

We thank Dr Alfonso Luque and Dr Luisa Iruela-Arispe (Molecular Biology Institute, UCLA) for assistance with antisera production, and Maria del Carmen Plaza for growth of cell lines in the early stages of this work. We are grateful to Kristina Johnson and Dr Michael Carey (Department of Biological Chemistry, UCLA) for reagents and helpful discussions about RNA polymerase II, and to Dr Alvaro Monteiro (Rockefeller University, NY, USA) for plasmids and discussions related to mammalian GAL4 transcrip-tion assays. We thank Jan Johnson for screening the bovine hybrid panels and Srinivas Kata (Texas A&M) for computer analysis of the radiation hybrid results. We also thank Amilcar Rizzo and Robert Lurvey for assistance in the production of GST fusion proteins, and the other members of the Lane lab for helpful discussions.The authors acknowledge support from an NRSA pre-doctoral fellow-ship (GM07185) to SAK, and support from the Ovarian Cancer Research Fund, the Stein-Oppenheimer Foundation, the Jennifer Jones Simon Foundation, the Cancer Research Coordinating Committee of the University of California, the Stop Cancer Foundation, and funding from the Department of Defense, Ovarian Cancer Initiative (OC98-0006) to TFL.

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Authors and Affiliations

  1. Molecular Biology Institute, UCLA, Los Angeles, 90095, CA, USA

    Susan A Krum & Timothy F Lane

  2. Texas A&M University, College Station, 77843, TX, USA

    James E Womack

  3. Departments of Obstetrics and Gynecology and Biological Chemistry, The David Geffen School of Medicine at UCLA, Los Angeles, 90095-1740, CA, USA

    Timothy F Lane

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Correspondence to Timothy F Lane.

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Krum, S., Womack, J. & Lane, T. Bovine BRCA1 shows classic responses to genotoxic stress but low in vitro transcriptional activation activity. Oncogene 22, 6032–6044 (2003). https://doi.org/10.1038/sj.onc.1206515

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