含有活化的组蛋白3赖氨酸4 (H3K4me3) 和抑制性赖氨酸27 (H3K27me3) 三甲基化修饰的双价表观遗传调控区域与关键发育基因相关。这些双价区域在缺少分化信号时抑制转录，但保持调控基因处于准备状态，以便及时激活。先前的研究表明，赛芘唑 (EPZ6438) 是一种组蛋白3赖氨酸27 (H3K27) 甲基转移酶抑制剂，可以抑制成骨分化，并且抑制赛芘唑有助于体外骨母细胞分化承诺和体内骨形成。在本研究中，我们探讨了 EPZ6438 的机制影响，EPZ6438 是FDA批准的治疗上皮肉瘤的 Ezh2 抑制剂，因此这种药物可能被重新应用于刺激骨形成的临床适应症治疗。我们发现，EPZ6438 减少了翼状骨合成和成熟所需的增强子上的 H3K27me3 标记，并刺激了MC3T3成骨前体细胞的成熟。此外，根据转录组（RNA-seq）和表观遗传组（ChIP-seq）数据，EPZ6438 激活了与无翼/整合（WNT）、腺苷酸环化酶（cAMP）和Hedgehog（Hh）信号通路相关的双价基因。利用选择性通路抑制剂和沉默RNA进行功能分析表明，WNT和Hh通路调节了Ezh2抑制后的成骨分化。引人注目的是，我们发现失去Hh响应的转录调节因子Gli1，但不是Gli2，与EPZ6438协同加速成骨细胞分化。这些研究确定了Ezh2、Hh-Gli1信号通路和双价调控基因在抑制骨形成中的表观遗传相互作用。我们的发现对于需要骨量累积和/或逆转骨质流失的促进骨形成的临床应用可能具有重要的转化意义。内容版权所有©2023年作者。Elsevier Inc. 出版。保留所有权利。

Bivalent epigenomic regulatory domains containing both activating histone 3 lysine 4 (H3K4me3) and repressive lysine 27 (H3K27me3) trimethylation are associated with key developmental genes. These bivalent domains repress transcription in the absence of differentiation signals but maintain regulatory genes in a poised state to allow for timely activation. Previous studies demonstrated that enhancer of zeste homolog 2 (Ezh2), a histone 3 lysine 27 (H3K27) methyltransferase, suppresses osteogenic differentiation and that inhibition of Ezh2 enhances commitment of osteoblast progenitors in vitro and bone formation in vivo. Here, we examined the mechanistic effects of Tazemetostat (EPZ6438), an FDA approved Ezh2 inhibitor for epithelioid sarcoma treatment, because this drug could potentially be repurposed to stimulate osteogenesis for clinical indications. We find that Tazemetostat reduces H3K27me3 marks in bivalent domains in enhancers required for bone formation and stimulates maturation of MC3T3 pre-osteoblasts. Furthermore, Tazemetostat activates bivalent genes associated with the Wingless/Integrated (WNT), Adenylyl cyclase (cAMP) and Hedgehog (Hh) signaling pathways based on transcriptomic (RNA-seq) and epigenomic (ChIP-seq) data. Functional analyses using selective pathway inhibitors and silencing RNAs demonstrate that the WNT and Hh pathways modulate osteogenic differentiation after Ezh2 inhibition. Strikingly, we show that loss of the Hh-responsive transcriptional regulator Gli1, but not Gli2, synergizes with Tazemetostat to accelerate osteoblast differentiation. These studies establish epigenetic cooperativity of Ezh2, Hh-Gli1 signaling, and bivalent regulatory genes in suppressing osteogenesis. Our findings may have important translational ramifications for anabolic applications requiring bone mass accrual and/or reverse bone loss.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.