Unbiased identification of signal-activated transcription factors by barcoded synthetic tandem repeat promoter screening (BC-STAR-PROM).
Unbiased identification of signal-activated transcription factors by barcoded synthetic tandem repeat promoter screening (BC-STAR-PROM).
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DOI：
10.1101/gad.284828.116
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发表时间：
2016-08-15
影响因子：
10.5
通讯作者：
Schibler U
中科院分区：
文献类型：
--
作者：
Gosselin P;Rando G;Fleury-Olela F;Schibler U
Gosselin等人设计了一种广泛适用的方法，称为BC-STAR-PROM，可以在不事先了解其性质的情况下识别信号激活的tf。为了验证BC-STAR-PROM的概念，他们将其应用于鉴定由影响肌动蛋白和微管蛋白细胞骨架动力学的药物诱导的tf。转录因子（TFs）在健康和疾病控制途径的发现是最重要的兴趣。我们设计了一种广泛应用的方法，称为条形码合成串联重复启动子筛选（BC-STAR-PROM），以识别信号激活的tf，而无需任何先验知识。BC-STAR-PROM文库由~ 3000个荧光素酶表达载体组成，每个载体都含有一个启动子（由6个合成随机DNA串联重复序列组成）和一个3 '未翻译mRNA区域内20个碱基对（bp）的相关条形码。总的来说，启动子序列包含了40万bp的随机DNA，这个序列的复杂性足以捕获大多数tf。用文库转染的细胞暴露在一个信号中，它编码的mrna通过下一代条形码测序进行计数。这允许在单个实验中同时跟踪每一个~ 3000个合成启动子的活性。本文通过将BC-STAR-PROM应用于影响肌动蛋白和微管蛋白细胞骨架动力学的药物诱导的tf的鉴定，建立了BC-STAR-PROM的概念证明。BC-STAR-PROM显示血清反应因子（SRF）是肌动蛋白聚合和微管解聚诱导的唯一直接早期TF。已知细胞骨架动力学的这种变化发生在细胞分裂周期中，实时生物发光显微镜确实揭示了增殖细胞中细胞自主srf -心肌素相关TF （MRTF）活性的发作。
Gosselin et al. designed a widely applicable method, dubbed BC-STAR-PROM, to identify signal-activated TFs without any prior knowledge of their properties. To establish proof of concept for BC-STAR-PROM, they applied it to the identification of TFs induced by drugs affecting actin and tubulin cytoskeleton dynamics. The discovery of transcription factors (TFs) controlling pathways in health and disease is of paramount interest. We designed a widely applicable method, dubbed barcorded synthetic tandem repeat promoter screening (BC-STAR-PROM), to identify signal-activated TFs without any a priori knowledge about their properties. The BC-STAR-PROM library consists of ∼3000 luciferase expression vectors, each harboring a promoter (composed of six tandem repeats of synthetic random DNA) and an associated barcode of 20 base pairs (bp) within the 3′ untranslated mRNA region. Together, the promoter sequences encompass >400,000 bp of random DNA, a sequence complexity sufficient to capture most TFs. Cells transfected with the library are exposed to a signal, and the mRNAs that it encodes are counted by next-generation sequencing of the barcodes. This allows the simultaneous activity tracking of each of the ∼3000 synthetic promoters in a single experiment. Here we establish proof of concept for BC-STAR-PROM by applying it to the identification of TFs induced by drugs affecting actin and tubulin cytoskeleton dynamics. BC-STAR-PROM revealed that serum response factor (SRF) is the only immediate early TF induced by both actin polymerization and microtubule depolymerization. Such changes in cytoskeleton dynamics are known to occur during the cell division cycle, and real-time bioluminescence microscopy indeed revealed cell-autonomous SRF–myocardin-related TF (MRTF) activity bouts in proliferating cells.
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影响因子：
9.2
作者：
Bershadsky, A;Chausovsky, A;Geiger, B
通讯作者：
Geiger, B
影响因子：
2.3
作者：
Sage D;Unser M;Salmon P;Dibner C
通讯作者：
Dibner C
DOI：
10.1007/978-1-60761-376-3_4
发表时间：
2009-01-01
期刊：
CYTOSKELETON METHODS AND PROTOCOLS
影响因子：
--
作者：
Holzinger, Andreas
通讯作者：
Holzinger, Andreas
影响因子：
3.7
作者：
Reddy P;Deguchi M;Cheng Y;Hsueh AJ
通讯作者：
Hsueh AJ
影响因子：
5.3
作者：
Rivas, FV;O'Keefe, JP;Gajewski, TF
通讯作者：
Gajewski, TF