Development of a multiple reaction monitoring (MRM)-based LC-MS/MS method for the quantification of post-translational modifications on histone H3 variants in Arabidopsis thaliana†

Abstract

Background: although the canonical histone H3.1 and its variant H3.3 differ by only four amino acids, they exhibit distinct genome-wide binding patterns and regulate different biological pathways. Post-translational modifications (PTMs) on histone tails mediate diverse downstream regulatory processes, raising the question of whether H3.1 and H3.3 harbor variant-specific modifications. However, the minimal amino acid differences between H3.1 and H3.3 make it challenging to distinguish and quantify them using traditional methods. Results: in this study, we developed an integrated multiple reaction monitoring (MRM)-based LC-MS/MS method to accurately differentiate and quantify K27 and K36 modifications on H3.1 and H3.3 in Arabidopsis thaliana. Our findings show that H3.1 contains more K27 methylation marks, associated with gene silencing, whereas H3.3 is enriched in K36 methylation, a mark of active transcription. Additionally, we compared K36 methylation levels in wild-type and SDG8-depleted cells, revealing that the K36 methyltransferase SDG8 shows a strong preference for H3.3 in both in vitro and in vivo assays. By analyzing public datasets, we further identified a strong correlation between H3.3 and the regions where H3K36me3 levels were reduced in sdg8 knockout cells. Significance: the MRM-based LC-MS/MS method established in this study provides a reliable and robust tool for the quantification of histone H3.1 and H3.3 PTMs in Arabidopsis thaliana. We demonstrate that the methyltransferase SDG8 shows a strong substrate preference for H3.3. This discovery highlights the importance of histone variant-specific modifications and suggests new avenues for research into their regulatory roles.