乙酰甲基赖氨酸:用于标记染色质的一种新的翻译后修饰

Hua Guo, Fangfang Zhou, Long Zhang
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Acetylation neutralizes histone's positive charge, weakening the DNA–histone interaction for easier binding with transcription factors. Unlike acetylation, methylation affects reader protein binding and leads to changes in chromatin structure, resulting in transcription suppression or activation.<span><sup>2</sup></span> Although it is commonly believed that acetylation and monomethylation are mutually exclusive modifications on a single residue, chemical principles permit a lysine residue to be stably acetylated and monomethylated to create a tertiary amide, Kacme.</p><p>To provide evidence for the existence of Kacme in cellular proteins, researchers synthesized Fmoc–Lys (Ac, Me)-OH as a building block to create a central Kacme residue peptide library and used them as an antigen to generate a specific antiserum against Kacme.<span><sup>1</sup></span> Kacme antisera demonstrated high specificity toward Kacme peptides but not otherwise identical Kac, Kme1, and propionyllysine (Kpr). 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This discovery offers a novel avenue for investigation of chromatin biology (Figure 1).</p><p>Histones play a crucial role in regulating gene expression and chromatin structure through PTMs such as acetylation (Kac) and methylation (Kme), impacting transcriptional activity. Acetylation neutralizes histone's positive charge, weakening the DNA–histone interaction for easier binding with transcription factors. 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引用次数: 0

摘要

最近,发表在《自然》(Nature)上的一项由 Lu-Culligan 等人进行的研究提出了 Nε-acetyl-Nε -methyllysine (Kacme),即甲基化和乙酰化发生在组蛋白 H4 上赖氨酸的同一个侧链上,是组蛋白 H4 上的一种细胞翻译后修饰 (PTM)。组蛋白通过乙酰化(Kac)和甲基化(Kme)等 PTM 在调节基因表达和染色质结构方面发挥着至关重要的作用,从而影响转录活性。乙酰化中和了组蛋白的正电荷,削弱了 DNA 与组蛋白的相互作用,从而更容易与转录因子结合。与乙酰化不同,甲基化会影响阅读蛋白的结合,导致染色质结构发生变化,从而抑制或激活转录。尽管人们通常认为乙酰化和单甲基化是对单个残基的相互排斥的修饰,但化学原理允许一个赖氨酸残基被稳定地乙酰化和单甲基化,从而产生一个三级酰胺--Kacme。为了提供细胞蛋白质中存在 Kacme 的证据,研究人员合成了 Fmoc-Lys (Ac, Me)-OH 作为构建模块,创建了一个中心 Kacme 残基肽库,并将其用作抗原,生成针对 Kacme 的特异性抗血清1。Kacme 抗血清对 Kacme 多肽具有高度特异性,而对其他相同的 Kac、Kme1 和丙酰基甘氨酸(Kpr)则没有特异性。利用这种抗血清,研究人员分析了果蝇、小鼠和人类细胞系中细胞内的 Kacme 修饰,并确定组蛋白 H4 Lys5 和 Lys12 是人类细胞中的 Kacme 修饰位点。为了通过一种独立于抗血清的方法确认 Kacme 修饰,作者对合成的 H4K5acme 肽进行了同位素标记,以获得不同的离子诊断峰,然后再进行细胞内蛋白质组分析,这进一步证实了组蛋白中存在 Kacme。染色质免疫沉淀测序(ChIP-seq)是研究多种转录因子和其他染色质相关蛋白与 DNA 之间相互作用的一种极为强大的工具。通过在果蝇和人体细胞中使用 Kacme 抗原进行 ChIP-seq 测序,作者发现 Kacme 显著富集在基因启动子周围,尤其是在高表达基因中,而且其定位与活跃的染色质修饰密切相关。随后,Lu-Culligan 等人进行了瞬时转录组延时测序以检测转录活性,并进行了启动延时测序以研究启动子近端暂停的动力学1,证实了 Kacme 与转录和转录启动之间的正相关性。Kacme 形式的赖氨酸残基会发生单甲基化和随后的乙酰化,从而将 Kacme 修饰与两种酶联系起来:赖氨酸甲基转移酶(KMT)和乙酰基转移酶(KAT)。通过分析以前的 ChIP-seq 数据,作者发现 KAT p300 能特异性地乙酰化 Kme1 以生成 Kacme,为这一酶解过程提供了大量证据。接下来,研究人员将重点放在消除 Kacme 修饰上。通过采用免疫印迹和蛋白质组学技术,作者观察到用组蛋白去乙酰化酶(HDAC)/锌水解酶抑制剂三氯司他丁 A 处理的细胞中 Kacme 信号增加。同时,他们还发现 HDAC1 和 HDAC3 能在体外清除 H4K5ac 上的乙酰基,但不能清除 H4K5acme 上的乙酰基。1Bromodomain-containing proteins (BRDs)是一种保守的蛋白质-蛋白质相互作用模块,可选择性地识别并结合乙酰化赖氨酸残基,尤其是组蛋白中的乙酰化赖氨酸残基,在调控基因表达方面起着至关重要的作用。在使用生物素化 Kacme 肽的提取物中,BRD2 与 BRD3 和 GAS41 被选择性地富集,这表明 Kacme 具有识别染色质蛋白并与之相互作用的能力。作者通过晶体结构和等温滴定量热分析证明,Kacme残基占据了BRD2上与Kac残基结合的相同结合袋,并确定天冬酰胺(N156)是Kacme和BRD2相互作用的关键残基。总之,甲基化和乙酰化发生在同一个赖氨酸残基上的新修饰为染色质生物学和相关疾病带来了机遇和挑战。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Acetyl-methyllysine: A new posttranslational modification used to mark chromatin

Acetyl-methyllysine: A new posttranslational modification used to mark chromatin

A recent study, conducted by Lu-Culligan et al., published in Nature, proposed Nε-acetyl-Nε -methyllysine (Kacme) that both methylation and acetylation occur on the same side chain of lysine as a cellular posttranslational modification (PTM) on histone H4.1 Kacme can be recognized and bound by the chromatin protein bromodomain-containing 2 (BRD2), associating with active chromatin marks and enhanced transcriptional initiation. This discovery offers a novel avenue for investigation of chromatin biology (Figure 1).

Histones play a crucial role in regulating gene expression and chromatin structure through PTMs such as acetylation (Kac) and methylation (Kme), impacting transcriptional activity. Acetylation neutralizes histone's positive charge, weakening the DNA–histone interaction for easier binding with transcription factors. Unlike acetylation, methylation affects reader protein binding and leads to changes in chromatin structure, resulting in transcription suppression or activation.2 Although it is commonly believed that acetylation and monomethylation are mutually exclusive modifications on a single residue, chemical principles permit a lysine residue to be stably acetylated and monomethylated to create a tertiary amide, Kacme.

To provide evidence for the existence of Kacme in cellular proteins, researchers synthesized Fmoc–Lys (Ac, Me)-OH as a building block to create a central Kacme residue peptide library and used them as an antigen to generate a specific antiserum against Kacme.1 Kacme antisera demonstrated high specificity toward Kacme peptides but not otherwise identical Kac, Kme1, and propionyllysine (Kpr). By utilizing this antiserum, researchers analyzed intracellular Kacme modifications in fruit fly, mouse, and human cell lines and identified histone H4 Lys5 and Lys12 as Kacme-modified sites in human cells. To confirm Kacme modification through an antisera-independent approach, the authors isotopically labeled the synthetic H4K5acme peptide to obtain distinct ion diagnostic peaks before conducting intracellular proteomic analysis, which further supported the presence of Kacme in histones.

Chromatin immunoprecipitation sequencing (ChIP-seq) is an extremely powerful tool for studying interactions between multiple transcription factors and other chromatin-associated proteins and DNA.3 By performing ChIP-seq with Kacme antisera in fruit flies and human cells, the authors found that Kacme was significantly enriched around gene promoters, especially in highly expressed genes, and its localization was strongly associated with active chromatin modifications. Subsequently, Lu-Culligan et al. conducted transient-transcriptome time-lapse sequencing to examine transcriptional activity, and start-time-lapse sequencing to investigate the kinetics of promoter–proximal pausing,1 confirming the positive correlation between Kacme and both transcription and transcription initiation.

The lysine residue in the Kacme form undergoes monomethylation and subsequent acetylation, linking Kacme modifications to two enzymes: lysine methyltransferases (KMT) and acetyltransferases (KAT). Through analysis of previous ChIP-seq data, the authors identified that KAT p300 specifically acetylates Kme1 to generate Kacme, providing substantial evidence for this enzymatic process. Consistent results were observed in cell lines treated with A-485 (a p300 inhibitor).

Next, researchers focused on eliminating the Kacme modifications. By employing immunoblotting and proteomic techniques, the authors observed an increased Kacme signal in cells treated with trichostatin A, an inhibitor of histone deacetylase (HDAC)/zinc hydrolase. Simultaneously, they discovered that HDAC1 and HDAC3 could remove the acetyl group from H4K5ac but not H4K5acme in vitro. These findings support the idea that Kacme shares chemical and functional properties with Kac; however, its susceptibility to scavenging reactions varies significantly.1

Bromodomain-containing proteins (BRDs) are conserved protein–protein interaction modules that selectively recognize and bind to acetylated lysine residues, particularly in histones, and play a crucial role in regulating gene expression.4 BRD2 was selectively enriched in extracts using biotinylated Kacme peptides, along with BRD3 and GAS41, indicating that Kacme has the ability to identify and interact with chromatin proteins. By the crystal structural and isothermal titration calorimetry analysis, the authors demonstrated that Kacme residues occupy the same binding pocket on BRD2 that has been shown to bind Kac residues and identified asparagine (N156) as a critical residue for Kacme and BRD2 interaction.

In conclusion, a new modification, in which both methylation and acetylation occur at the same lysine residue, raises both opportunities and challenges in chromatin biology and related disease. For example, targeting the different sensitivities of Kacme and Kac to deacetylase may offer new treatment strategies for deacetylase-related diseases. Moreover, the chemical characteristics of Kacme resemble those of neutral Kac, suggesting its involvement in nonhistone proteins, which are crucial in various cellular processes or may be related to diseases, reinforcing the importance of this novel modification. One limitation of studying Kacme is its interaction with Kac, Kme, and other factors that may cause interference and affect the reliability of experimental results during the analysis. This paper introduces novel concepts and methods for studying protein modifications by proposing reasonable hypotheses, followed by chemical biological synthesis of modified peptides and subsequent verification through biological experiments. Although these new ideas come with complex methods and technologies, continuous progress in science and technology will overcome these challenges.

Hua Guo wrote the manuscript and prepared the figure. Fangfang Zhou and Long Zhang provided valuable discussion. All authors have read and approved the article.

The authors declare no conflict of interest.

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