摘要
乳腺癌易感基因BRCA1突变引起的遗传性乳腺癌占 4 0 %～ 5 0 % ,其突变引起的遗传性乳腺癌和卵巢癌的比例至少为 80 %。许多乳腺癌易感突变发生在BRCA1C末端转录激活结构域 (15 6 0～ 186 3aa) ,但该区域大部分突变导致何种表型 (良性多态性或乳腺癌易感突变 )目前还不清楚。由于染色质结构调节是基因转录调节的早期事件 ,该文基于lac阻遏物识别和结合lac操纵基因的原理 ,利用染色质结构检测技术比较BRCA1转录激活结构域不同突变体与野生型的染色质伸展活性。将 1种野生型、2种良性多态型 (S16 13G和M16 5 2I)和 4种乳腺癌易感突变型(A170 8E、M1775R、W1837R和Y185 3term)转录激活区片段以正确相位融合于lac阻遏物的下游 ,得到野生型重组质粒pwt和pS16 13G、pM16 5 2I、pA170 8E、pM1775R、pW1837R及pY185 3term 6种突变型重组质粒。Westernblot检测表明 ,这些重组质粒分别转染AO3-1细胞后均表达了相应的融合蛋白。对这些重组质粒的染色质伸展活性检测表明 ,野生型pwt和两种良性多态性突变体不具有染色质伸展活性或只有极微弱的染色质伸展活性 ,而其他 4种乳腺癌易感突变体均具有过强的染色质伸展活性 ,提示利用染色质伸展技术可预测BRCA1转录激活区基因型与乳腺癌发生风险的表现型的关系。
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.
