Department of Chemistry, University of Konstanz; Universitätsstraße 10, 78464 Konstanz, Germany.
Konstanz Research School Chemical Biology, University of Konstanz; Universitätsstraße 10, 78464 Konstanz, Germany.
Nucleic Acids Res. 2024 Jan 11;52(1):101-113. doi: 10.1093/nar/gkad1113.
Post-translational modifications (PTMs) of histones have fundamental effects on chromatin structure and function. While the impact of PTMs on the function of core histones are increasingly well understood, this is much less the case for modifications of linker histone H1, which is at least in part due to a lack of proper tools. In this work, we establish the assembly of intact chromatosomes containing site-specifically ubiquitylated and acetylated linker histone H1.2 variants obtained by a combination of chemical biology approaches. We then use these complexes in a tailored affinity enrichment mass spectrometry workflow to identify and comprehensively characterize chromatosome-specific cellular interactomes and the impact of site-specific linker histone modifications on a proteome-wide scale. We validate and benchmark our approach by western-blotting and by confirming the involvement of chromatin-bound H1.2 in the recruitment of proteins involved in DNA double-strand break repair using an in vitro ligation assay. We relate our data to previous work and in particular compare it to data on modification-specific interaction partners of free H1. Taken together, our data supports the role of chromatin-bound H1 as a regulatory protein with distinct functions beyond DNA compaction and constitutes an important resource for future investigations of histone epigenetic modifications.
组蛋白的翻译后修饰 (PTMs) 对染色质结构和功能有根本影响。虽然 PTM 对核心组蛋白功能的影响越来越被理解，但连接组蛋白 H1 的修饰则要少得多，这在一定程度上是由于缺乏适当的工具。在这项工作中，我们通过化学生物学方法的组合，建立了含有特异性泛素化和乙酰化连接组蛋白 H1.2 变体的完整染色质小体的组装。然后，我们在定制的亲和富集质谱工作流程中使用这些复合物来鉴定和全面表征染色质小体特异性细胞相互作用组，并在全蛋白质组范围内研究特异性连接组蛋白修饰的影响。我们通过 Western-blotting 进行验证和基准测试，并通过体外连接测定证实染色质结合的 H1.2 参与招募涉及 DNA 双链断裂修复的蛋白质，从而验证了我们的方法。我们将我们的数据与以前的工作进行了比较，特别是与游离 H1 的修饰特异性相互作用伙伴的数据进行了比较。总之，我们的数据支持染色质结合的 H1 作为一种调节蛋白的作用，其功能不仅仅局限于 DNA 压缩，并为未来研究组蛋白表观遗传修饰构成了重要资源。
