Chemogenetic Minitool for Dissecting the Roles of Protein Phase Separation

Biomolecular condensate is an emerging structural entity that regulates various cellular processes. Recent studies have discovered the phase-separation (PS) capability of several transcription factors (TFs) including YAP/TAZ upon biological stimuli, which provide new mechanisms of gene regulation. However, it remains mostly unanswered as to whether PS from a diffuse state to a phase-separated state promotes gene transcription. To address this question, we have designed a chemogenetic tool, dubbed SPARK-ON, which manipulates the PS of YAP and TAZ without a biological stimulus, forming condensates that are transcriptionally active, containing the DNA-binding partner TEAD, genomic DNA, transcriptional machinery, and nascent RNA. Most importantly, PS of TAZ increases the transcription of its target genes. Therefore, our data indicate that PS promotes gene transcription of TAZ. SPARK-ON is advantageous to current mutagenesis-based approaches that are often problematic when mutagenesis affects the transcriptional activity of a TF. Furthermore, protein abundance levels also affect gene transcription, but PS depends on protein abundance because PS occurs only when the protein level is above a saturation concentration. SPARK-ON decouples PS from protein abundance levels without introducing mutations and thus will find important applications in understanding the biological roles of PS for many TFs and other biomolecular condensates.生物分子凝集体（biomolecular condensate）是一类新兴的结构实体，可调控多种细胞生理过程。近期研究发现，包括YAP/TAZ在内的多种转录因子（transcription factors，TFs）在生物刺激下具备相分离（phase-separation，PS）能力，这为基因调控机制研究提供了全新思路。然而，目前仍未明确的核心问题是：蛋白质从弥散状态转变为相分离状态的过程是否会促进基因转录。为解答这一问题，本研究设计了一款命名为SPARK-ON的化学遗传工具（chemogenetic tool），可在无需生物刺激的条件下调控YAP与TAZ的相分离过程，形成具有转录活性的凝集体。该凝集体包含DNA结合伴侣蛋白TEAD、基因组DNA、转录机器与新生RNA。尤为关键的是，TAZ的相分离可上调其靶基因的转录水平。据此，本研究数据证实相分离能够促进TAZ介导的基因转录。相较于当前主流的基于诱变的研究方法，SPARK-ON具有显著优势：后者常因诱变操作干扰转录因子的转录活性而存在固有缺陷。此外，蛋白质丰度水平同样会影响基因转录，但相分离过程本身依赖于蛋白质丰度：仅当蛋白质浓度超过饱和阈值时，相分离才会发生。SPARK-ON可在不引入突变的前提下，将相分离过程与蛋白质丰度水平解耦，因此该工具在阐明多种转录因子及其他生物分子凝集体的相分离生物学功能方面，具有重要的应用前景。