Braun Simon M G, Kirkland Jacob G, Chory Emma J, Husmann Dylan, Calarco Joseph P, Crabtree Gerald R
Departments of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, CA, 94305, USA.
Department of Chemical Engineering, Stanford University, Stanford, CA, 94305, USA.
Nat Commun. 2017 Sep 15;8(1):560. doi: 10.1038/s41467-017-00644-y.
Understanding the causal link between epigenetic marks and gene regulation remains a central question in chromatin biology. To edit the epigenome we developed the FIRE-Cas9 system for rapid and reversible recruitment of endogenous chromatin regulators to specific genomic loci. We enhanced the dCas9-MS2 anchor for genome targeting with Fkbp/Frb dimerizing fusion proteins to allow chemical-induced proximity of a desired chromatin regulator. We find that mSWI/SNF (BAF) complex recruitment is sufficient to oppose Polycomb within minutes, leading to activation of bivalent gene transcription in mouse embryonic stem cells. Furthermore, Hp1/Suv39h1 heterochromatin complex recruitment to active promoters deposits H3K9me3 domains, resulting in gene silencing that can be reversed upon washout of the chemical dimerizer. This inducible recruitment strategy provides precise kinetic information to model epigenetic memory and plasticity. It is broadly applicable to mechanistic studies of chromatin in mammalian cells and is particularly suited to the analysis of endogenous multi-subunit chromatin regulator complexes.Understanding the link between epigenetic marks and gene regulation requires the development of new tools to directly manipulate chromatin. Here the authors demonstrate a Cas9-based system to recruit chromatin remodelers to loci of interest, allowing rapid, reversible manipulation of epigenetic states.
理解表观遗传标记与基因调控之间的因果联系仍然是染色质生物学的核心问题。为了编辑表观基因组，我们开发了FIRE-Cas9系统，用于将内源性染色质调节因子快速且可逆地招募到特定基因组位点。我们通过Fkbp/Frb二聚化融合蛋白增强了用于基因组靶向的dCas9-MS2锚定物，以实现化学诱导所需染色质调节因子的接近。我们发现，招募mSWI/SNF（BAF）复合物足以在几分钟内对抗多梳蛋白，从而导致小鼠胚胎干细胞中二价基因转录的激活。此外，将Hp1/Suv39h1异染色质复合物招募到活跃启动子上会沉积H3K9me3结构域，导致基因沉默，在洗去化学二聚体后这种沉默可以逆转。这种可诱导的招募策略为模拟表观遗传记忆和可塑性提供了精确的动力学信息。它广泛适用于哺乳动物细胞中染色质的机制研究，尤其适用于对内源性多亚基染色质调节复合物的分析。理解表观遗传标记与基因调控之间的联系需要开发新工具来直接操纵染色质。在此，作者展示了一种基于Cas9的系统，可将染色质重塑因子招募到感兴趣的位点，从而实现对表观遗传状态的快速、可逆操纵。
