JGG 2002, 29(11): 0-948 DOI:   ISSN: 1673-8527 CN: 11-5450/R           

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Studies of Mutations in BRCA1 Transactivation Domain by Visulization of Chromatin Structure

YE Qi-Nong1; HU Yan-Fen2; ZHONG Hong-Jun1; LI Rong2; HUANG Cui-Fen1

1.Beijing Institute of Biotechnology, Beijing 100850,China 2.Department of Biochemistry and Molecular Genetics, University of Virginia, VA 22908,USA

Abstract

Mutations in breast cancer susceptibility gene 1 (BRCA1) account for approximately 40% ~ 50% of familial breast cancer cases and for more than 80% of inherited breast and ovarian cancer cases. Many cancer-predisposing mutations are located in the C-terminal region that functions as a transcriptional activation domain, but most of the mutations in the transactivation domain identified to date cannot be readily distinguished as either disease-associated mutations or benign polymorphisms. Because chromatin structure regulation is an early event in gene transcription control, the chromatin unfolding activities of different transactivation domain variants were compared with that of the wild-type transactivation domain by use of an approach that allows visualization of large-scale chromatin structure through lac repressor/lac operator recognition. To do this, different constructs of the transactivation domain were selected as follows:(a) the wild-type transactivation domain;(b) two polymorphisms (S1613G and M1652I); and (c) four cancer-predisposing mutations (A1708E, M1775R, W1837R and Y1853term). All of the constructs were made by fusing in frame with lac repressor. Western blot analysis indicated that all of the fusion proteins were expressed in AO3_ 1 cells, in which multiple copies of the lac operator were integrated to produce a heterochromatic region of the genome. The chromatin unfolding assay showed that ,like the wild-type transactivation domain, two variants that represent benign polymorphisms did not induce chromatin unfolding or only induced subtle change. Contrary to the behaviors of the wild type and two benign variants, four cancer-predisposing mutations in the transactivation domain superactivate the chromatin unfolding. The results suggest that the chromatin unfolding assay can aid in the characterization of deleterious mutations in the C-terminal transactivation domain of BRCA1 and may provide more reliable presymptomatic risk assessment.

Keywords BRCA1   breast cancer   chromatin   transcription activation  
Received 1900-01-01 Revised 1900-01-01 Online 2002-11-10 
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