Differentially Isotope‐Labeled Nucleosomes To Study Asymmetric Histone Modification Crosstalk by Time‐Resolved NMR Spectroscopy

Post‐translational modifications (PTMs) of histones regulate chromatin structure and function. Because nucleosomes contain two copies each of the four core histones, the establishment of different PTMs on individual “sister” histones in the same nucleosomal context, that is, asymmetric histone PTMs, are difficult to analyze. Here, we generated differentially isotope‐labeled nucleosomes to study asymmetric histone modification crosstalk by time‐resolved NMR spectroscopy. Specifically, we present mechanistic insights into nucleosomal histone H3 modification reactions in cis and in trans, that is, within individual H3 copies or between them. We validated our approach by using the H3S10phK14ac crosstalk mechanism, which is mediated by the Gcn5 acetyltransferase. Moreover, phosphorylation assays on methylated substrates showed that, under certain conditions, Haspin kinase is able to produce nucleosomes decorated asymmetrically with two distinct types of PTMs.     

Development of a DNA‐Templated Peptide Probe for Photoaffinity Labeling and Enrichment of the Histone Modification Reader Proteins

Histone post‐translational modifications (HPTMs) provide signal platforms to recruit proteins or protein complexes to regulate gene expression. Therefore, the identification of these recruited partners (readers) is essential to understand the underlying regulatory mechanisms. However, it is still a major challenge to profile these partners because their interactions with HPTMs are rather weak and highly dynamic. Herein we report the development of a HPTM dual probe based on DNA‐templated technology and a photo‐crosslinking method for the identification of HPTM readers. By using the trimethylation of histone H3 lysine 4, we demonstrated that this HPTM dual probe can be successfully utilized for labeling and enrichment of HPTM readers, as well as for the discovery of potential HPTM partners. This study describes the development of a new chemical proteomics tool for profiling HPTM readers and can be adapted for broad biomedical applications.     

基于细胞红外光谱预测乙酰化细胞的辐射敏感性

组蛋白乙酰化是表观遗传修饰的一种重要方式。肿瘤细胞的组蛋白大部分呈现低乙酰化状态,而组蛋白去乙酰化酶抑制剂（histone deacetylase inhibitor,HDACi）可以增加肿瘤细胞的乙酰化水平,诱导细胞周期阻滞及凋亡。曲古菌素A （trichostatin A,TSA）是组蛋白去乙酰化酶抑制剂的代表药物之一,能够提高肿瘤细胞组蛋白和非组蛋白的乙酰化水平。傅里叶变换红外（Fourier Transform Infrared,FTIR）光谱可以对无染色、无标记的生物样品进行无损检测,具有特征性明显、快速、分辨率高、重复性好等优点,已被广泛用于细胞的微观生物过程的研究。本文利用红外光谱技术结合免疫荧光技术的手段,研究TSA处理细胞后的乙酰化作用效果,发现红外光谱中甲基与亚甲基的伸缩振动强度之比能够表征细胞内的乙酰化水平变化,然后基于红外光谱的分析结果预测了乙酰化状态不同的细胞辐射敏感性的变化。结果表明,乙酰化细胞的辐射损伤效应可以通过甲基与亚甲基的伸缩振动强度之比进行评价,且该比值与细胞的辐射敏感性呈正相关,表明红外光谱技术可以辅助预测细胞的辐射敏感性,并进行细胞表观遗传学特征与辐射效应关系的研究。Histone acetylation is one of important epigenetic modifications, and histone in most of tumor cells shows low acetylation state. However, histone deacetylase inhibitor (HDACi) can correct abnormal acetylation status, induce cell cycle arrest and apoptosis. Trichostatin A (TSA) is one of the representatives of histone deacetylase inhibitors, which can inhibit histone deacetylase, increase the acetylation level of histone and nonhistone in cell. Fourier transform infrared (FTIR) spectroscopy is a powerful analytical tool which can detect nondestructively, quatitatively and quantitatively biological samples without bio-tagging and bio-labeling. FTIR spectroscopy technology has multiple advantages, including finger-print characteristics, rapid analysis, high resolution and good repeatability. Therefore, it has been widely used in the research of biological processes. This work applied FTIR spectroscopy to study the changes in cells treated with TSA, compared the acetylation level according to FTIR intensity ratio of methyl to methylene stretching vibration, and based on the FTIR analysis predicted the radiosensitivity of the cells with different acetylation levels. As a result, we have verified that the damage caused by radiation in acetylated cells can be evaluated by the ratio of methyl and methylene intensity which is positively correlated with cellular radiosensitivity. Therefore, this work demonstrates that FTIR spectroscopy can be useful for the prediction of radiosensitivity and may also open a door for the study of relationship between epigenetics and radiation bio-effects.     

Expansion of the lysine acylation landscape

Leaving marks: the number of known posttranslational modifications for lysine has been expanded considerably. In addition to acetylation of side-chain amino functionalities of lysine residues in proteins, crotonylation, succinylation, and malonylation have now been identified as posttranslational modifications in histone and in non-histone proteins.