A high-throughput method to identify trans-activation domains within transcription factor sequences.
A high-throughput method to identify trans-activation domains within transcription factor sequences.
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DOI：
10.15252/embj.201798896
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发表时间：
2018-08-15
期刊：
影响因子：
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通讯作者：
Stark A
中科院分区：
文献类型：
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作者：
Arnold CD;Nemčko F;Woodfin AR;Wienerroither S;Vlasova A;Schleiffer A;Pagani M;Rath M;Stark A
尽管转录因子（TF）是基因调控的核心参与者，并已被广泛研究，但其调节性反式激活结构域（tAD）通常仍然未知，并且缺乏tAD的系统功能表征。在这里，我们提出了一种新的高通量方法tAD-seq，用于并行测试来自不同TF的数千个候选tAD。我们通过合并筛选鉴定的tAD在单独的荧光素酶测定中验证，而中性区域则没有。有趣的是，在TF序列内的任意位置发现tAD，并且可以含有氨基酸（例如，谷氨酰胺）重复区或重叠结构域，包括通常注释为DNA结合的螺旋-环-螺旋结构域。我们还鉴定了非天然阅读框架中的tAD，证实了随机序列可以作为tAD发挥作用，尽管作用很弱。识别tAD作为短的蛋白质序列足以转录激活将使TF功能的系统研究，这特别是对于不同的转录激活功能的TF仍然知之甚少。
Even though transcription factors (TFs) are central players of gene regulation and have been extensively studied, their regulatory trans‐activation domains (tADs) often remain unknown and a systematic functional characterization of tADs is lacking. Here, we present a novel high‐throughput approach tAD‐seq to functionally test thousands of candidate tADs from different TFs in parallel. The tADs we identify by pooled screening validate in individual luciferase assays, whereas neutral regions do not. Interestingly, the tADs are found at arbitrary positions within the TF sequences and can contain amino acid (e.g., glutamine) repeat regions or overlap structured domains, including helix–loop–helix domains that are typically annotated as DNA‐binding. We also identified tADs in the non‐native reading frames, confirming that random sequences can function as tADs, albeit weakly. The identification of tADs as short protein sequences sufficient for transcription activation will enable the systematic study of TF function, which—particularly for TFs of different transcription activating functionalities—is still poorly understood.