Molecular interactions between Fanconi anemia pathway and familial breast cancer pathway mediated by BCCIP.

范可尼贫血（Fanconi anemia， FA）途径与家族性乳腺癌易感基因（familial breast cancer susceptibility gene， BRCA）途径之间的功能联系已有报道，但这些相互作用在体内的生理意义和确切的分子机制尚未明确。以鸡（ch） FancD2的n端片段为诱饵进行酵母双杂交（Y2H）筛选，鉴定出brca2相互作用蛋白chBCCIP的c端片段。为了证实这种相互作用，我们用表达his标记的全长chBCCIP和tap标记的全长chFancD2的质粒转染了293T细胞。TAP-FancD2下拉和anti-His Western blotting可以成功检测到这种相互作用。据报道，人类BCCIP与BRCA2相互作用。为了确认FancD2-BCCIP-BRCA2相互作用，我们使用人类cdna进行了Y2H分析，但没有一个组合显示出显著的相互作用。然后，我们使用鸡的cdna进行哺乳动物双杂交（M2H）试验。我们克隆了全长和原始片段，在载体上进行了Y2H筛选，但我们没有检测到任何显著的相互作用。chBRCA2片段的进一步表达对这些相互作用没有影响。这些结果表明，BCCIP和BRCA2之间的相互作用不稳定或不稳定。尽管我们分析了数百个克隆，但在鸡DT40细胞中建立BCCIP基因条件靶向的尝试尚未成功。由于酵母BCIP基因是脊椎动物BCIP的同源基因，因此在DT40细胞中缺失BCCIP可能导致死亡。我们也尝试用RNA干扰（RNAi）的方法敲低人细胞中的BCCIP基因。由于GFP-human BCCIP在293T细胞中通过共转染RNAi显著降低了表达水平，我们现在研究了降低内源性BCCIP基因表达的合适条件。在我们的相关研究中，我们使用双敲除DT40细胞报道了FANCC和BRCA2基因在细胞生长和x射线敏感性方面的显形。这些结果表明FA和BRCA通路在DT40细胞中具有遗传相互作用。有必要进行进一步调查。少

Functional linkage between Fanconi anemia (FA) pathway and familial breast cancer susceptibility gene (BRCA) pathway has been reported, but physiological significance and exact molecular mechanism of these interactions in vivo have not been identified. By performing yeast two-hybrid (Y2H) screening using N-terminal fragment of chicken (ch) FancD2 as a bait, we have identified C-terminal fragment of chBCCIP, a BRCA2-interacting protein. To confirm this interaction, we transfected 293T cells with plasmids, which express His-tagged full-length chBCCIP and TAP-tagged full-length chFancD2. TAP-FancD2 pull-down and anti-His Western blotting could successfully detect this interaction. Human BCCIP was reported to interact with BRCA2. To confirm FancD2-BCCIP-BRCA2 interaction, we performed Y2H analysis using human cDNAs, but none of the combinations showed significant interaction. Then, we performed mammalian two-hybrid (M2H) assay using chicken cDNAs. We cloned full-length and original fragmen … More ts used in Y2H screening in the vectors, but we could not detect any significant interactions tested combinations. Further expression of chBRCA2 fragments did not affect to these interactions. These results suggested that interactions between BCCIP and BRCA2 were not stable and/or very week. Attempt for establishing the conditional targeting of BCCIP gene in chicken DT40 cells has not been successful, even though we analyzed several hundred clones. Since yeast BCIP gene, a homolog of vertebrate BCCIP, is an essential gene, BCCIP deletion in DT40 cell could lead to lethality. We also tried to knock down the BCCIP gene in human cells by RNA interference (RNAi) method. Since expression levels of GFP-human BCCIP was markedly reduced by co-transfected RNAi in 293T cells, we have now examined the suitable conditions for reduction of endogenous BCCIP gene expression. In our related study, we reported that FANCC and BRCA2 gene epistatic regarding cell growth and sensitivity to X-ray using double knock out DT40 cells. These results indicated that FA and BRCA pathways interact genetically in DT40 cells. Further investigations would be warranted. Less