The N-terminal conserved domain of the Arabidopsis H2B variant HTB8 binds peptidyl prolyl cis-trans isomerase (PPI) and is required for localization at the centromeric region

The eleven HTB variants found in Arabidopsis are divided into three classes, with class III containing the single HTB8 variant that is highly divergent from the other HTB proteins. It consists of an extended N-terminal region having a conserved domain (HTB8-CD) with the core sequence of KVVxETVxVxV, which is found within the N-terminal extension of all HTB8 orthologs in angiosperms. Pull-down assay with GST-CD immobilized onto glutathione beads followed by proteome analysis revealed two major proteins bound to the CD, namely, HTB3 and peptidyl prolyl cis-trans isomerase (PPI). The latter is an enzyme that catalyzes the cis-trans isomerization of peptide bonds N-terminal to proline residues in polypeptide chains, and we found it is localized to the nucleus. We generated transgenic Arabidopsis plants expressing HTB8-GFP, HTB5-GFP (class II), and HTB9-GFP (class I) under the control of the CaMV 35S promoter and inspected their subnuclear localization under a confocal microscope. In all cell types examined, HTB8-GFP showed unique localization in the nucleus, which differs from the localization pattern of the other HTB variants, where it was restrictively localized at chromocenters. Expression of HTB8-GFP in Arabidopsis protoplasts showed similar centromeric localization as in transgenic plants, while deletion of the CD disrupted HTB8 nuclear localization. Thus, HTB8 lacking the CD (HTB8ΔCD-GFP) showed essentially even distribution within the nucleus, suggesting that the CD is essential for localization at heterochromatic chromocenters. The interplay between HTB8 and PPI is currently under study.