Genetic dissection of the BRCA2 promoter and transcriptional impact of DNA variants
Genetic dissection of the BRCA2 promoter and transcriptional impact of DNA variants
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DOI：
10.1007/s10549-018-4826-7
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发表时间：
2018-08-01
影响因子：
3.8
通讯作者：
Velasco, Eladio A.
中科院分区：
文献类型：
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作者：
Fraile-Bethencourt, Eugenia;Valenzuela-Palomo, Alberto;Velasco, Eladio A.
启动子突变可能影响转录，并可能与人类疾病有关。然而，乳腺癌（BC）基因的启动子并没有定期筛查。我们的目标是研究BRCA2启动子，以研究转录受损与疾病之间可能的相关性。对95例高危BC患者BRCA2基因近端启动子和核心启动子进行了测序。生成brca2启动子插入片段[转录起始位点(TSS) - 938 ~ + 312]，并将其克隆到萤火虫荧光素酶载体pGL4.10中。启动子变异和缺失通过定点诱变引入，并通过双荧光素酶测定和半定量RT-PCR进行定量。在高危BC患者中检测到三种不同的变异：rs3092989、rs206118和rss563971900。13个重叠缺失的功能定位显示了4个下调片段（TSS位置）：- 59_10del /A mu del3（占野生型构建体活性的16%），- 104_55del /A mu del4 (62%), - 239_190del /A mu del7 (39%), - 464_415 /A mu del12(78%)，表明其中存在假定的转录激活子基序。此外，6个微缺失导致荧光素酶过表达：+32_+81del/A mu del1（356%）、-14_+36del/A mu del2（180%）、-194_-145del/A mu del6（154%）、-284_-235del/A mu del8（168%）、-329_-280del/A mu del9（111%）和-509_-460del/A mu del13(139%)，这表明抑制因子存在。15个启动子变异（包括在患者中检测到的）的功能分析显示，其中10个显著改变了表达，7个上调（113-163%），3个下调（rs551887850_G、rss570548398_t、rs55880202_T; 72-83%）。其中8个位于ENCODE-DNase Hypersensitive Cluster （TSS - 185至+ 105）中，已知大多数活性转录基序位于此。BRCA2表达对启动子变异高度敏感，因为大多数启动子都会引起相关变化。此外，我们绘制了BRCA2启动子的关键区域，这些区域可能构成调控变异的潜在靶点。三个snp适度降低荧光素酶活性，但其潜在致病性的确认需要进一步分析。这些数据加强了筛选乳腺癌基因启动子区域的需要，以期发现新的有害突变。
Promoter mutations may affect transcription and can be associated with human diseases. However, the promoters of the breast cancer (BC) genes are not regularly screened. Our goal was to investigate the BRCA2 promoter in order to study a possible correlation between impaired transcription and disease.The proximal and core promoter of the BRCA2 gene was sequenced in 95 high-risk BC patients. A BRCA2-promoter insert [- 938 to + 312 from the transcription start site (TSS)] was generated and cloned into the firefly luciferase vector pGL4.10. Promoter variants and deletions were introduced by site-directed mutagenesis and quantified by Dual-Luciferase assays and semi-quantitative RT-PCR.Three different variants were detected in high-risk BC patients: rs3092989, rs206118, and rs563971900. Functional mapping of 13 overlapping deletions revealed four down-regulating segments (TSS positions): -59_-10del/A mu del3 (16% of activity of the wild-type construct), -104_-55del/A mu del4 (62%), -239_-190del/A mu del7 (39%), -464_-415/A mu del12 (78%), suggesting the presence therein of putative transcriptional activator motifs. Additionally, six microdeletions rendered luciferase overexpression: +32_+81del/A mu del1 (356%), -14_+36del/A mu del2 (180%), -194_-145del/A mu del6 (154%), -284_-235del/A mu del8 (168%), -329_-280del/A mu del9 (111%), and -509_-460del/A mu del13 (139%), which is indicative of repressor elements. Functional assays of 15 promoter variants (including those detected in patients) showed that ten of them significantly altered expression with seven up-regulating (113-163%) and three down-regulating (rs551887850_G, rs570548398_T, rs55880202_T; 72-83%) SNPs. Eight of them were located in an ENCODE-DNase Hypersensitive Cluster (TSS - 185 to + 105) where most active transcriptional motifs are known to be placed.BRCA2 expression is highly sensitive to promoter variations as most of them induced relevant changes. Moreover, we mapped critical regions of the BRCA2 promoter that may constitute potential targets for regulatory variants. Three SNPs moderately decreased luciferase activity, but confirmation of its potential pathogenicity requires further analysis. These data reinforce the need to screen the promoter regions of breast cancer genes with a view to discovering novel deleterious mutations.