Metabolites such as short-chain fatty acids can interact with chromatin and affect transcription by modifying histone proteins. The variety of histone modifications makes it technically hard to delineate their function in a site- and modification-type-specific way. Qin et al. developed a single-site-resolved multi-omics (SiTomics) strategy based on genetic code expansion technology to profile the interacting proteomic and genomic landscape for a given histone modification in cells. The authors first used an unbiased proteomics approach to identify histone-modification sites in response to short-chain fatty acids, revealing known modification sites (such as K56) and new sites (K36, K37 and K64) on histone H3. To characterize these modifications under physiological conditions, the authors used genetic code expansion to incorporate lysine analogs that carry a given modification into a specific site of histone H3 to generate pre-modified histones in living cells, which could then be integrated into chromatin for genomic location reading via next-generation sequencing. The photoaffinity moiety in the lysine analogs enables the formation of covalent crosslinking with proximal proteins after light illumination, and the crosslinked proteins can be identified through mass spectrometry. Using their SiTomics strategy, the authors showed that unique modifications even at the same histone site can have diverse interactomes and gene-regulation functions. For example, H3K56 crotonylation showed enrichment in promoter regions and exhibited a cooperative effect on facilitating nucleosomal DNA accessibility with the active histone mark H3K4me3. H3K56 β-hydroxybutrylation can be enriched at super-enhancers, along with H3K27acetylation and transcriptional coactivators, resulting in looser 3D chromatin organization. These results indicate that SiTomics is a powerful platform for elucidating site-specific functions of histone modifications in a physiologically relevant environment.

短链脂肪酸等代谢物可与染色质相互作用，并通过修饰组蛋白影响转录。组蛋白修饰的多样性使得在技术上难以以位点和修饰类型特定的方式描述它们的功能。秦等。开发了一种基于遗传密码扩展技术的单点解析多组学（SiTomics）策略，以分析细胞中给定组蛋白修饰的相互作用蛋白质组学和基因组学景观。作者首先使用无偏蛋白质组学方法识别响应短链脂肪酸的组蛋白修饰位点，揭示组蛋白 H3 上的已知修饰位点（如 K56）和新位点（K36、K37 和 K64）。为了表征生理条件下的这些修饰，作者使用遗传密码扩展将携带给定修饰的赖氨酸类似物掺入组蛋白 H3 的特定位点，以在活细胞中生成预修饰的组蛋白，然后可以将其整合到染色质中，通过下一代测序读取基因组位置。赖氨酸类似物中的光亲和部分能够在光照后与近端蛋白质形成共价交联，并且可以通过质谱法鉴定交联的蛋白质。使用他们的 SiTomics 策略，作者表明即使在同一组蛋白位点的独特修饰也可以具有不同的相互作用组和基因调控功能。例如，H3K56 巴豆酰化显示启动子区域富集，并与活性组蛋白标记 H3K4me3 在促进核小体 DNA 可及性方面表现出协同作用。H3K56 β-羟基丁酰化可在超增强子处富集，连同 H3K27 乙酰化和转录共激活因子，导致 3D 染色质结构松散。这些结果表明，SiTomics 是一个强大的平台，可用于阐明生理相关环境中组蛋白修饰的位点特异性功能。