Molecular mechanisms of functional phase separation in eukaryotic gene transcription

点击翻译按钮获取中文摘要

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.