Histone Deubiquitination Assay in Nicotiana benthamiana

Histone modifications are a group of post-translational modifications on histones which can alter chromatin structure and affect gene expression. Histone ubiquitination is a histone modification found in particular on histone H2A and H2B. Histone ubiquitination can be reversed by ubiquitin-specific proteases (UBP). Here, we describe an in vivo assay for histone deubiquitination activity. After infiltrating UBP12 into Nicotiana benthamiana leaves, H2Aub was visualized by immunocytochemistry. Nicotiana benthamiana leaves, which show high agro infiltration efficiency, were used for transient UBP12 expression for a labor- and time-saving protocol. Reduced H2Aub levels indicated histone deubiquitination activity of UBP12. The clear visualization of nuclei of N. benthamiana leaves makes this method able to easily measure the level of histone modification in vivo by using specific antibodies, providing robust clues of protein function. Thus, this protocol is a powerful complementation to in vitro assays of histone deubiquitination activity.