Specific tracking of N-terminal clipping on histone H3 in Tetrahymena enabled by a custom branched-peptide antibody
摘要
Histone H3 clipping, a unique but evolutionarily conserved post-translational modification that irreversibly removes the N-terminal tail of H3, has been reported across diverse eukaryotic lineages. In Tetrahymena thermophila, a ciliate with nuclear dimorphism, H3 clipping is a bona fide proteolytic event generating H3F (H3-Fast) by removing the first six N-terminal amino acids, specifically in the transcriptionally silent micronucleus (MIC). However, the detection of H3F remains technically demanding, time-consuming, and lacks spatio-temporal resolution. To overcome this, a 2 × branched peptide antigen was developed to generate a high-specificity antibody that exclusively recognizes H3F, effectively distinguishing it from full-length H3 and other truncation variants. This antibody eliminated the need for labor-intensive MIC isolation and histone extraction, enabling rapid, small-scale detection directly from whole-cell lysates. Using this antibody, dynamic subcellular localization of H3F was investigated through different cell stages, revealing its persistence during vegetation, starvation and early conjugation. However, H3F disappeared concurrently with macronuclear anlage formation, supporting the notation that removal of H3F is a prerequisite for the new macronucleus development. Comparative analyses further revealed that H3 Ser10 phosphorylation, though previously used as an alternative H3F marker, actually occurs strictly after clipping, refining the temporal hierarchy of these two chromatin events. This work provides the first in situ, high-resolution method to track endogenous H3 clipping, providing both a technical platform and new biological insight into the developmental regulation of proteolytic histone modifications.