Molecular mechanisms of functional phase separation in eukaryotic gene transcription

RNA聚合酶II （Pol II）对蛋白质编码基因的转录在细胞分化和机体发育过程中受到高度调控。在人类细胞中，Pol II转录受约1600个转录因子调控，这些转录因子结合靶基因附近的特定DNA序列，并包含通常在转录激活中起作用的低复杂性区域（Lambert et al., 2018）。转录因子如何与Pol II机制相互作用仍然知之甚少，但最近的证据表明，液-液相变可能是这种相互作用的基础（Chong et al., 2018; Hnisz et al., 2017; Kato and McKnight, 2017）。事实上，我们最近表明，Pol II的高度保守的c -末端结构域（CTD）可以在人类细胞中相分离和介导Pol II酶的聚集（boeing等）。这些结果为使用综合实验-理论方法分析转录因子如何与Pol II相互作用提供了一个很好的起点。首先，我们将系统地分析转录因子和共激活因子中的内在无序区（idr）序列，以获得可能相互作用并因此相互相分离的idr类别（Coordinator: Soeding）。其次，我们将从每个类别中制备示例性重组转录因子，并分析这些转录因子是否可以进行相分离，以及它们是否可以包含在相分离的Pol II CTD液滴中（协调器：Cramer）。第三，我们将通过核磁共振分析相分离CTD和转录因子样品的残留结构，针对相互作用的分子基础（Coordinator: Zweckstetter）。我们的努力将阐明相分离现象及其对基因转录的功能重要性，并将解决不同的转录因子如何与相同的一般转录机制相互作用的谜题。我们的研究将为spp2191的总体目标做出重大贡献，揭示相分离驱动的分子机制和生理功能。

Transcription of protein-coding genes by RNA polymerase II (Pol II) is highly regulated during cell differentiation and organismal development. In human cells, Pol II transcription is regulated by ~1,600 transcription factors, which bind specific DNA sequences in the vicinity of target genes and contain low-complexity regions that often function in transcription activation (Lambert et al., 2018). It remains poorly understood how transcription factors interact with the Pol II machinery, but recent evidence suggests that liquid-liquid phase transitions may underlie such interactions (Chong et al., 2018; Hnisz et al., 2017; Kato and McKnight, 2017). Indeed we recently showed that the highly conserved C-terminal domain (CTD) of Pol II can phase separate and mediate the clustering of Pol II enzymes in human cells (Boehning et al.). These results provide a great starting point for analyzing how transcription factors interact with Pol II, using an integrated experimental-theoretical approach. First, we will systematically analyze the sequences of intrinsically disordered regions (IDRs) in transcription factors and coactivators to obtain classes of IDRs that are likely to interact and thus to phase-separate with each other (Coordinator: Soeding). Second, we will prepare exemplary recombinant transcription factors from each class and analyze whether these can undergo phase separation and whether they can be included into phase-separated Pol II CTD droplets (Coordinator: Cramer). Third, we will analyze residual structure in phase-separated CTD and transcription factor samples by nuclear magnetic resonance, aiming at the molecular basis of the interactions (Coordinator: Zweckstetter). Our efforts will elucidate the phenomenon of phase separation and its functional importance for gene transcription, and will address the enigmatic question of how the different transcription factors can interact with the same general transcription machinery. Our studies will contribute significantly to the overarching goal of SPP 2191, to unravel the molecular mechanisms and physiological functions that are driven by phase separation.