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

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 cycleregulated 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) (bBRCA ). Interestingly, when tested in the GAL4 transcriptional activation assay, fulllength 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.

BRCA1 Associates with Processive RNA Polymerase II

“…This panel is informative, since mouse and human BRCA1 genes share only 60% amino acid identity (40), whereas cow and dog BRCA1 are ϳ80% identical to each other and to both mouse and human proteins (39). Initially, regions homologous to amino acids 1380 -1863 of human BRCA1 were tested in the MCF7 one-hybrid assay (Figs.…”

“…BRCA1 from human cell extracts were identified with either Ab4 (Oncogene Research Products, San Diego, CA) or C-20 (Santa Cruz Biotechnology). BRCA1 from bovine cells was detected with C3F (39). Antisera to FCP1 were generously provided by Dr. Michael E. Dahmus (University of California, Davis).…”

“…The murine BRCA1-CTD (codons 1334 -1813) was then amplified with the primers 5Ј-CCAAGAATTCC-CGGATACGAGAGTGAAACAA-3Ј and 5Ј-AAGCACCAAAGACTGAG-A-3Ј (40). Bovine BRCA1-CTD and full-length constructs were described previously (39). All PCR fragments were ligated into pCR-blunt II TOPO (Invitrogen) and sequenced in both directions to confirm identity with previously reported sequences.…”

Direct Stimulation of Transcription Initiation by BRCA1 Requires Both Its Amino and Carboxyl Termini

“…However, in a defined system assayed in vitro, a Gal4 fusion to the carboxyl terminus of BRCA1 activates transcription, independent of other activators (9), suggesting an intrinsic transcriptional activity for BRCA1. A subsequent study found that Gal4 fusions to full-length BRCA1 could not activate transcription in transfected cells and that the degree of transcriptional activation conferred by Gal4 fusions to the carboxyl terminus of bovine BRCA1 was much lower than human BRCA1 (10). Since the human carboxyl terminus is more acidic than the bovine version, the transcriptional activity may simply be a function of its acidity.…”

Expression Patterns of the BRCA1 Splicing Variants in Canine Normal Tissues and Mammary Gland Tumors

“…RT reactions were carried out using d(T) [12][13][14][15][16][17][18] (GE Healthcare) and ReverTra Ace (TOYOBO) following the manufacturer's directions. Five µg of total RNA from each sample was used as template for each reaction, and 5 µl of cDNA from each sample was used for PCR.…”