In contrast to CUT&Tag approaches for profiling bulk histone modifications, current CUT&Tag methods for analysing specific transcription factor (TF)-DNA interactions remain technically challenging due to TFs having relatively low abundance. Moreover, an efficient CUT&Tag strategy for plant TFs is not yet available. Here, we first applied biotinylated Tn5 transposase-mediated CUT&Tag (B-CUT&Tag) to produce high-quality libraries for interrogating TF-DNA interactions. B-CUT&Tag combines streptavidin-biotin-based DNA purification with routine CUT&Tag, optimizing the removal of large amounts of intact chromatin not targeted by specific TFs. The biotinylated chromatin fragments are then purified for construction of deep sequencing libraries or qPCR analysis. We applied B-CUT&Tag to probe genome-wide DNA targets of Squamosa promoter-binding-like protein 9 (SPL9), a well-established TF in Arabidopsis; the resulting profiles were efficient and consistent in demonstrating its well-established target genes in juvenile-adult transition/flowering, trichome development, flavonoid biosynthesis, wax synthesis and branching. Interestingly, our results indicate functions of AtSPL9 in modulating growth-defence trade-offs. In addition, we established a method for applying qPCR after CUT&Tag (B-CUT&Tag-qPCR) and successfully validated the binding of SPL9 in Arabidopsis and PHR2 in rice. Our study thus provides a convenient and highly efficient CUT&Tag strategy for profiling TF-chromatin interactions that is widely applicable to the annotation of cis-regulatory elements for crop improvement.

中文翻译：

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生物素化 Tn5 转座酶介导的 CUT&Tag 有效描述植物中的转录因子-DNA 相互作用

与用于分析大量组蛋白修饰的 CUT&Tag 方法相比，当前用于分析特定转录因子 (TF)-DNA 相互作用的 CUT&Tag 方法在技术上仍然具有挑战性，因为 TF 的丰度相对较低。此外，目前还没有针对植物 TF 的有效 CUT&Tag 策略。在这里，我们首先应用生物素化的 Tn5 转座酶介导的 CUT&Tag (B-CUT&Tag) 来生成用于询问 TF-DNA 相互作用的高质量文库。B-CUT&Tag 将基于链霉亲和素-生物素的 DNA 纯化与常规 CUT&Tag 相结合，优化去除大量未被特定 TF 靶向的完整染色质。然后纯化生物素化的染色质片段，用于构建深度测序文库或 qPCR 分析。我们应用了 B-CUT&拟南芥; 由此产生的概况在证明其在幼年-成年过渡/开花、毛状体发育、类黄酮生物合成、蜡合成和分枝方面的成熟目标基因方面是有效且一致的。有趣的是，我们的结果表明 AtSPL9 在调节增长防御权衡中的功能。此外，我们建立了一种在 CUT&Tag (B-CUT&Tag-qPCR) 之后应用 qPCR 的方法，并成功验证了拟南芥中 SPL9与水稻中 PHR2的结合。因此，我们的研究为分析 TF-染色质相互作用提供了一种方便且高效的 CUT&Tag 策略，该策略广泛适用于作物改良的顺式调控元件的注释。