Cryo-EM uncovers polycomb interactions Polycomb family enzymes include the chromatin modifiers PRC1 and PRC2, which are involved in gene repression. Although the catalytic functions of these complexes are well known, their functional relationship is not. Kasinath et al. used cryo–electron microscopy (cryo-EM) to visualize the interactions between nucleosomes containing ubiquitinated histone H2A, the product of PRC1, and the PRC2-activating cofactors JARID2 and AEBP2, providing the molecular basis for PRC1-dependent recruitment of PRC2. They also show that JARID2 and AEBP2 partially overcome the inhibitory effect of PRC2 by two trimethyl lysine transcription marks on histones. This work suggests that PRC2 regulation involves an intricate interplay between PRC2 cofactors and histone posttranslational modifications. Science, this issue p. eabc3393 Cryo-EM analysis reveals polycomb factor activities and cross-talk involved in gene repression. INTRODUCTION Histone modification activity of the polycomb repressive complexes 1 and 2 (PRC1 and PRC2) is critical for the establishment and maintenance of gene expression patterns and, thus, to the maintenance of cell identity. Distinct classes of cofactor proteins are known to regulate the functional activity and interplay of these two complexes, but we presently lack a comprehensive, mechanistic understanding of this process. Furthermore, PRC2 cofactors like AEBP2 and JARID2 also play a role in mediating the cross-talk between different histone posttranslational modifications and PRC2 recruitment and activity—a function that is important for the regulated control of gene expression. RATIONALE PRC1 is an E3 ubiquitin ligase responsible for the monoubiquitination of histone H2A (H2AK119ub1), a histone mark recognized by PRC2 and linked to its genomic recruitment. We used cryo–electron microscopy (cryo-EM) and biochemical activity assays to probe the role played by PRC2 cofactors JARID2 and AEBP2 in the recognition of H2AK119ub1 and the regulation of PRC2 activity. We extended our cryo-EM and biochemical activity analysis to examine the possible role played by JARID2 and AEBP2 in the cross-talk between the histone H3K4me3, H3K36me3 modifications linked to transcriptionally active regions, and PRC2 activity. RESULTS We find that JARID2 recognizes both the ubiquitin moiety in H2AK119ub1 and the conserved histone H2A-H2B acidic patch. We also observe that the tandem zinc fingers of AEBP2 interact with ubiquitin and the histone H2A-H2B surface on the other side of the nucleosome. Biochemical assays show a secondary activation of PRC2 by JARID2 and AEBP2 on H2AK119ub1-containing nucleosomes besides the primary EED-mediated allosteric activation of PRC2 by methylated JARID2. Furthermore, we also find that the joint presence of JARID2 and AEBP2 partially reduces the inhibition of PRC2 methyltransferase activity by the transcriptionally active histone posttranslational modifications H3K4me3 and H3K36me3. Cryo-EM visualization of PRC2 that contains JARID2 and AEBP2 interacting with a H3K4me3-containing nucleosome shows the coexistence of states in which the histone H3 tail is either absent or engaged and reaching the catalytic site in PRC2, which provides a physical basis for the partial activity of the complex on H3K4me3-containing nucleosomes. CONCLUSION Our studies indicate that cofactors JARID2 and AEBP2 play a crucial role in both the recruitment and activation of PRC2 through their recognition of H2AK119ub1, which is generated by PRC1. Additionally, our work suggests that JARID2 and AEBP2 are likely to play a key role in regulating PRC2 activity on genomic regions with active transcription marks. The examination of the genomic distribution in embryonic stem cells of PRC2 core proteins together with JARID2 and AEBP2 will be important to further define their role in the tight regulation of PRC2 activity. Regulation of PRC2 methyltransferase activity by cofactors JARID2 and AEBP2 and by histone posttranslational modification. Although core PRC2 is a weak enzyme, it is allosterically activated by JARID2 and AEBP2. The presence of monoubiquitinated histone H2A, the product of PRC1 activity, is recognized by both JARID2 and AEBP2 through interactions that likely mediate recruitment of PRC2 to polycomb sites in the genome and further activate the methyltransferase activity of PRC2 on K27 of histone H3. Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) cooperate to determine cell identity by epigenetic gene expression regulation. However, the mechanism of PRC2 recruitment by means of recognition of PRC1-mediated H2AK119ub1 remains poorly understood. Our PRC2 cryo–electron microscopy structure with cofactors JARID2 and AEBP2 bound to a H2AK119ub1-containing nucleosome reveals a bridge helix in EZH2 that connects the SET domain, H3 tail, and nucleosomal DNA. JARID2 and AEBP2 each interact with one ubiquitin and the H2A-H2B surface. JARID2 stimulates PRC2 through interactions with both the polycomb protein EED and the H2AK119-ubiquitin, whereas AEBP2 has an additional scaffolding role. The presence of these cofactors partially overcomes the inhibitory effect that H3K4me3 and H3K36me3 exert on core PRC2 (in the absence of cofactors). Our results support a key role for JARID2 and AEBP2 in the cross-talk between histone modifications and PRC2 activity.

中文翻译：

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JARID2 和 AEBP2 在 H2AK119ub1 和其他组蛋白修饰存在的情况下调节 PRC2

Cryo-EM 揭示了 polycomb 相互作用 Polycomb 家族酶包括参与基因抑制的染色质修饰剂 PRC1 和 PRC2。虽然这些配合物的催化功能是众所周知的，但它们的功能关系却不是。卡西纳特等人。使用低温电子显微镜（cryo-EM）来观察含有泛素化组蛋白 H2A（PRC1 的产物）的核小体与 PRC2 激活辅因子 JARID2 和 AEBP2 之间的相互作用，为 PRC1 依赖性募集 PRC2 提供了分子基础。他们还表明，JARID2 和 AEBP2 通过组蛋白上的两个三甲基赖氨酸转录标记部分克服了 PRC2 的抑制作用。这项工作表明PRC2 调控涉及PRC2 辅因子和组蛋白翻译后修饰之间复杂的相互作用。科学，本期第 3 页。eabc3393 Cryo-EM 分析揭示了与基因抑制有关的多梳因子活性和串扰。引言 polycomb 抑制复合物 1 和 2（PRC1 和 PRC2）的组蛋白修饰活性对于基因表达模式的建立和维持至关重要，因此对于细胞特性的维持也是如此。已知不同类别的辅因子蛋白可调节这两种复合物的功能活性和相互作用，但我们目前对该过程缺乏全面的机械理解。此外，像AEBP2和JARID2这样的PRC2辅助因子也在介导不同组蛋白翻译后修饰与PRC2募集和活性之间的串扰中发挥作用——这一功能对于基因表达的调节控制很重要。RATIONALE PRC1 是一种 E3 泛素连接酶，负责组蛋白 H2A (H2AK119ub1) 的单泛素化，这是一种被 PRC2 识别并与其基因组募集相关的组蛋白标记。我们使用低温电子显微镜（cryo-EM）和生化活性测定来探测PRC2辅因子JARID2和AEBP2在识别H2AK119ub1和调节PRC2活性中所起的作用。我们扩展了我们的冷冻电镜和生化活性分析，以检查 JARID2 和 AEBP2 在组蛋白 H3K4me3、与转录活性区域相关的 H3K36me3 修饰和 PRC2 活性之间的串扰中可能发挥的作用。结果 我们发现 JARID2 识别 H2AK119ub1 中的泛素部分和保守的组蛋白 H2A-H2B 酸性斑块。我们还观察到 AEBP2 的串联锌指与核小体另一侧的泛素和组蛋白 H2A-H2B 表面相互作用。生化分析显示 JARID2 和 AEBP2 在含有 H2AK119ub1 的核小体上对 PRC2 的二次激活，除了由甲基化 JARID2 对 PRC2 的初级 EED 介导的变构激活之外。此外，我们还发现 JARID2 和 AEBP2 的共同存在部分降低了转录活性组蛋白翻译后修饰 H3K4me3 和 H3K36me3 对 PRC2 甲基转移酶活性的抑制。含有 JARID2 和 AEBP2 的 PRC2 与含有 H3K4me3 的核小体相互作用的低温 EM 可视化显示了组蛋白 H3 尾不存在或接合并到达 PRC2 中的催化位点的状态共存，这为复合物在含有 H3K4me3 的核小体上的部分活性提供了物理基础。结论 我们的研究表明，辅因子 JARID2 和 AEBP2 通过识别由 PRC1 产生的 H2AK119ub1 在 PRC2 的募集和激活中起关键作用。此外，我们的工作表明 JARID2 和 AEBP2 可能在调节具有活性转录标记的基因组区域的 PRC2 活性方面​​发挥关键作用。检查PRC2核心蛋白与JARID2和AEBP2在胚胎干细胞中的基因组分布对于进一步确定它们在PRC2活性的严格调节中的作用非常重要。辅因子 JARID2 和 AEBP2 以及组蛋白翻译后修饰对 PRC2 甲基转移酶活性的调节。虽然核心PRC2是一种弱酶，它由 JARID2 和 AEBP2 变构激活。单泛素化组蛋白 H2A（PRC1 活性的产物）的存在被 JARID2 和 AEBP2 通过相互作用识别，这些相互作用可能介导 PRC2 募集到基因组中的多梳位点并进一步激活 PRC2 在组蛋白 H3 的 K27 上的甲基转移酶活性。Polycomb 抑制复合物 1 和 2（PRC1 和 PRC2）通过表观遗传基因表达调控合作确定细胞身份。然而，通过识别PRC1介导的H2AK119ub1来招募PRC2的机制仍然知之甚少。我们的 PRC2 低温电子显微镜结构，辅因子 JARID2 和 AEBP2 与含有 H2AK119ub1 的核小体结合，揭示了 EZH2 中连接 SET 结构域、H3 尾和核小体 DNA 的桥螺旋。JARID2 和 AEBP2 各自与一种泛素和 H2A-H2B 表面相互作用。JARID2 通过与多梳蛋白 EED 和 H2AK119-泛素的相互作用刺激 PRC2，而 AEBP2 具有额外的支架作用。这些辅因子的存在部分克服了 H3K4me3 和 H3K36me3 对核心 PRC2 的抑制作用（在没有辅因子的情况下）。我们的结果支持 JARID2 和 AEBP2 在组蛋白修饰和 PRC2 活性之间的串扰中的关键作用。