Plant-specific HDT family histone deacetylases are nucleoplasmins.

Histone acetyltransferase (HAT)- and histone deacetylase (HDAC)-mediated histone acetylation and deacetylation regulate nucleosome dynamics and gene expression. HDACs are classified into different families, with HD-tuins or HDTs being specific to plants. HDTs show some sequence similarity to nucleoplasmins, the histone chaperones that aid in binding, storing, and loading H2A/H2B dimers to assemble nucleosomes. Here, we solved the crystal structure of the N-terminal domain (NTD) of all four HDTs (HDT1, HDT2, HDT3, and HDT4) from Arabidopsis (Arabidopsis thaliana). The NTDs form a nucleoplasmin fold, exist as pentamers in solution, and are resistant to protease treatment, high temperature, salt, and urea conditions. Structurally, HDTs do not form a decamer, unlike certain classical nucleoplasmins. The HDT-NTD requires an additional A2 acidic tract C-terminal to the nucleoplasmin domain for interaction with histone H3/H4 and H2A/H2B oligomers. We also report the in-solution structures of HDT2 pentamers in complex with histone oligomers. Our study provides a detailed structural and in vitro functional characterization of HDTs, revealing them to be nucleoplasmin family histone chaperones. The experimental confirmation that HDTs are nucleoplasmins may spark new interest in this enigmatic family of proteins.

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays.

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.