Regulation of the Dot1 histone H3K79 methyltransferase by histone H4K16 acetylation.
Regulation of the Dot1 histone H3K79 methyltransferase by histone H4K16 acetylation.
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DOI：
10.1126/science.abc6663
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发表时间：
2021-01-22
期刊：
影响因子：
--
通讯作者：
Armache KJ
中科院分区：
文献类型：
--
作者：
Valencia-Sánchez MI;De Ioannes P;Wang M;Truong DM;Lee R;Armache JP;Boeke JD;Armache KJ
核小体是染色质的主要重复单元，在包装和保护基因组的同时，也通过组蛋白的翻译后修饰传递各种调节信号。这些组蛋白修饰（例如，乙酰化，泛素化或甲基化）影响转录，复制，重组和修复等关键过程，通常形成复杂的网络，以确保染色质酶的精细调节信号。其中一种酶——进化上保守的端粒沉默干扰物（Dot1）——催化组蛋白H3赖氨酸79 （H3K79）的单甲基化、二甲基化和三甲基化。Dot1介导的H3K79甲基化是反组蛋白串扰的一个突出例子，在这个过程中，一个组蛋白及其修饰影响另一个组蛋白的修饰。在哺乳动物中，人类同源基因Dot1L在胚胎发生和造血中起着关键作用。尽管最近的进展已经提供了通过组蛋白H2B泛素化对Dot1L刺激的见解，但其他修饰如何机制地调节Dot1活性尚不清楚。我们对组蛋白赖氨酸乙酰化如何促进染色质酶的调节特别感兴趣。赖氨酸乙酰化在染色质去浓缩、转录激活和常染色质的维持中起着关键作用，在真核生物中是一种通用的抗沉默标记。在这里，我们提出了机制研究，表明组蛋白乙酰化如何调节Dot1的活性。组蛋白H4乙酰化、组蛋白H2B泛素化和H3甲基化是保守的共转录组蛋白修饰，它们共同确保转录过程中染色质结构的适当调节。这些修饰和存放它们的酶之间的串扰机制对于理解转录至关重要。低温电子显微镜（cryo-EM）技术的进步，特异乙酰化和泛素化核小体的能力，以及体外和体内实验的建立，使我们能够确定酵母通过组蛋白乙酰化和泛素化调控Dot1的详细机制。我们在体外测试了组蛋白H4不同乙酰化状态的核小体，我们发现H4的乙酰化对Dot1有变构刺激，并且这种作用是赖氨酸16 （H4K16ac）特异性的。其他已知的组蛋白H4乙酰化靶点（H4K5ac、H4K8ac和H4K12ac）不会刺激Dot1的活性，这凸显了H4K16乙酰化在调节染色质结构中的独特作用。我们还发现，H4K16乙酰化的影响是直接的，并被H2B泛素化（H2BUb）进一步增强，从而导致了Dot1的最佳催化速率。为了深入了解H4K16ac的刺激机制及其与H2BUb的协同作用，我们确定了两种低温电镜结构：一种是Dot1复合物，核小体同时携带H4K16ac和H2BUb，另一种是Dot1，核小体只携带H2BUb。通过检查我们的冷冻电镜数据集，Dot1与含有未乙酰化H4的核小体结合，将颗粒分为两个主要的三维（3D）类别。在第一类中，Dot1以催化构象与核小体结合。在第二类中，它以非催化构象结合。这与Dot1与H4K16乙酰化核小体结合的数据集不同。当H4K16ac存在时，低温电镜数据更加均匀，Dot1主要以催化构象结合到核小体上。这表明H4尾部的乙酰化限制了Dot1的采样空间，从而导致活性构象增加。因此，我们认为H2BUb部分限制了酵母Dot1在核小体上的构象，而H4K16ac进一步限制和稳定了活性构象。比较这两种低温电镜结构使我们能够确定对H4K16ac和H2BUb刺激Dot1至关重要的残基。位点定向诱变的Dot1结合核小体酶分析揭示了这些界面的细节。这些结果表明，H4K16ac和H2BUb对Dot1的变构刺激在H3K79二甲基化和三甲基化中起着至关重要的作用。这项工作证明了Dot1是如何通过组蛋白乙酰化调节的，以及H4K16ac如何与H2BUb协同调节Dot1。H4K16ac通过对抗沉默蛋白的结合，在打开染色质结构中发挥关键作用，同时刺激一种对转录很重要的酶。我们提供了一个例子，其中基本甲基转移酶Dot1的活性是通过不同组蛋白修饰之间的串扰来调节的，以确保表观遗传状态的最佳维持和繁殖。像这样的串扰可能代表了染色质酶的一般特性。H4K16乙酰化和H2BK123泛素化可以变构刺激Dot1对H3K79的甲基化。不同组蛋白翻译后修饰之间的串扰可以协调不同的染色质状态来调节转录和基因沉默。组蛋白H3赖氨酸79 （H3K79）甲基转移酶Dot1（端粒沉默的干扰物-1）在整个进化过程中是保守的，在人类白血病中发现了它的解除。在这里，我们提供的证据表明，组蛋白H4的乙酰化以一种不同于但与组蛋白H2B泛素化（H2BUb）协调的方式变构刺激酵母Dot1。我们进一步证明，这种刺激作用是特定于赖氨酸16 （H4K16ac）的乙酰化，这是染色质结构的中心修饰。我们提供了这种组蛋白串扰的机制，并表明H4K16ac和H2BUb在体外和体内H3K79二甲基化和三甲基化中起关键作用。这些数据揭示了控制H3K79甲基化的机制，并展示了H4K16ac、H3K79me和H2BUb如何共同调节基因转录和基因沉默，以确保表观遗传状态的最佳维持和繁殖。
Nucleosomes—the primary, repeating unit of chromatin—package and protect the genome while also transmitting various regulatory signals through, in part, posttranslational modifications of histones. These histone modifications (for example, acetylation, ubiquitination, or methylation) affect critical processes such as transcription, replication, recombination, and repair, often forming complicated networks to ensure finely tuned signaling for chromatin enzymes. One such enzyme—evolutionarily conserved disruptor of telomeric silencing (Dot1)—catalyzes mono-, di-, and trimethylation of histone H3 lysine 79 (H3K79). H3K79 methylation by Dot1 is a prominent example of trans-histone cross-talk, a process in which one histone and its modification affects the modification of another histone. In mammals, the human homolog Dot1L plays critical roles in embryogenesis and hematopoiesis. Although recent advances have provided insights into Dot1L stimulation through histone H2B ubiquitination, how other modifications mechanistically regulate Dot1 activity is not known. We were particularly interested in how histone lysine acetylation contributes to the regulation of chromatin enzymes. Lysine acetylation plays pivotal roles in chromatin decondensation, transcriptional activation, and maintenance of euchromatin, serving as a general antisilencing mark in eukaryotes. Here, we present mechanistic studies that show how histone acetylation regulates the activity of Dot1. Histone H4 acetylation, histone H2B ubiquitination, and H3 methylation are conserved cotranscriptional histone modifications that work together to ensure the appropriate regulation of chromatin structure during transcription. The mechanisms of cross-talk between these modifications and enzymes that deposit them are crucial for understanding transcription. Advances in cryo–electron microscopy (cryo-EM), the ability to make specifically acetylated and ubiquitinated nucleosomes, and established in vitro and in vivo assays allowed us to determine the detailed mechanisms of yeast Dot1 regulation through histone acetylation and ubiquitination. We tested nucleosomes with different acetylation states of histone H4 in vitro, and we show that Dot1 is allosterically stimulated by acetylation of H4 and that this effect is specific to lysine 16 (H4K16ac). The other known acetylation targets on histone H4 (H4K5ac, H4K8ac, and H4K12ac) do not stimulate the activity of Dot1, which highlights the distinctive role of H4K16 acetylation in regulating chromatin structure. We also show that the effect of H4K16 acetylation is direct and further enhanced by H2B ubiquitination (H2BUb), resulting in an optimal catalytic rate for Dot1. To gain mechanistic insights into stimulation by H4K16ac and its coordination with H2BUb, we determined two cryo-EM structures: one of Dot1 in complex, with nucleosomes bearing both H4K16ac and H2Bub, and the second of Dot1, with a nucleosome bearing only H2BUb. Upon examining our cryo-EM dataset of Dot1 bound to the nucleosome containing unacetylated H4, the particles classified into two main three-dimensional (3D) classes. In the first class, Dot1 is bound to the nucleosome in a catalytic conformation. In the second class, it is bound in a noncatalytic conformation. This is different from the dataset in which Dot1 is bound to an H4K16 acetylated nucleosome. When H4K16ac is present, the cryo-EM data is more homogeneous, and Dot1 is bound to the nucleosome predominantly in a catalytic conformation. This suggests a model in which acetylation of the H4 tail restricts the sampling space of Dot1, resulting in an active conformation leading to increased activity. We therefore propose that H2BUb partially restricts the conformation of yeast Dot1 on the nucleosome and that H4K16ac further restricts and stabilizes the active conformation. Comparing both of these cryo-EM structures allowed us to identify residues that are critical for Dot1 stimulation by H4K16ac and H2BUb. Site-directed mutagenesis of Dot1 coupled with enzymatic assays on nucleosomes revealed the details of these interfaces. These results show that the allosteric stimulation of Dot1 by H4K16ac and H2BUb plays a crucial role in H3K79 di- and trimethylation. This work demonstrates how Dot1 is regulated by histone acetylation and how H4K16ac coordinates with H2BUb to regulate Dot1. H4K16ac plays a critical role in opening chromatin structure by counteracting the binding of silencing proteins, while simultaneously stimulating an enzyme that is important for transcription. We provide an example in which the activity of the fundamental methyltransferase Dot1 is modulated through cross-talk between distinct histone modifications to ensure optimal maintenance and propagation of an epigenetic state. Cross-talk such as this may represent a general property of chromatin enzymes. H3K79 methylation by Dot1 is allosterically stimulated by H4K16 acetylation and by H2BK123 ubiquitination. Cross-talk between different histone posttranslational modifications can orchestrate distinct chromatin states to regulate transcription and gene silencing. Dot1 (disruptor of telomeric silencing-1), the histone H3 lysine 79 (H3K79) methyltransferase, is conserved throughout evolution, and its deregulation is found in human leukemias. Here, we provide evidence that acetylation of histone H4 allosterically stimulates yeast Dot1 in a manner distinct from but coordinating with histone H2B ubiquitination (H2BUb). We further demonstrate that this stimulatory effect is specific to acetylation of lysine 16 (H4K16ac), a modification central to chromatin structure. We provide a mechanism of this histone cross-talk and show that H4K16ac and H2BUb play crucial roles in H3K79 di- and trimethylation in vitro and in vivo. These data reveal mechanisms that control H3K79 methylation and demonstrate how H4K16ac, H3K79me, and H2BUb function together to regulate gene transcription and gene silencing to ensure optimal maintenance and propagation of an epigenetic state.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
16.8
作者：
Frederiks, Floor;Tzouros, Manuel;van Leeuwen, Fred
通讯作者：
van Leeuwen, Fred
影响因子：
16
作者：
Altaf, Mohammed;Utley, Rhea T.;Cote, Jacques
通讯作者：
Cote, Jacques
影响因子：
9.2
作者：
Feng, Q;Wang, HB;Zhang, Y
通讯作者：
Zhang, Y
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH