Structural and functional effects of histone variants on the nucleosome core particle

Abstract

DNA in eukaryotes is packed in association with a roughly equal mass of histone proteins to form a nucleo-protein complex known as chromatin. The fundamental unit of chromatin is the nucleosome core particle, which consists of two copies each of the four core histones H2A, H2B, H3 and H4 wrapped ~1.65 times by ~146 base pairs of DNA. In addition to compaction of DNA, the nucleosome core particle also plays an important role in regulating gene expression. It is the primary determinant of DNA accessibility in key cellular processes such as transcription, replication and repair. Several covalent modifications of the core histones are known to influence the structure and function of a nucleosome. However replacing one or more of the core histones with the corresponding histone variants can also vary the biochemical composition of the nucleosome. MacroH2A is a histone H2A variant that preferentially localizes to the inactive X-chromosome of adult female mammals. Unlike other core histones, macroH2A has a tripartite structural organization that consists of a histone domain and a non-histone domain connected by a linker domain. We have studied the structural and functional consequences of incorporation of the histone domain of macroH2A into nucleosome core particles. We show that the nucleosome structure remains unaffected by the sequence changes in most regions of the histone fold. The L1-loop of macroH2A is the only region that shows considerable structural differences compared to that of major type H2A. We show that this region is also responsible for the anomalies in the biochemical behavior of nucleosome core particles containing macroH2A (macro-NCPs). It has also been established in studies inspired by our structural and biochemical insights that the L1-loop is sufficient for the in vivo targeting of macroH2A. We have determined the 1.6Å crystal structure of the non-histone domain of macroH2A. We find that it is an α/β fold that contains a 7 stranded β-sheet and 5 α-helices. It is also remarkably devoid of distinct features on the surface of the protein and the charge distribution is unusually neutral for a chromatin-associated protein. We have also investigated the stoichiometry of nucleosomes containing different histone variants. We show that different histone variants show different propensities to form hybrid nucleosomes (nucleosomes containing one variant and one non-variant histone). MacroH2A in fact 'prefers' to form hybrid nucleosomes in vitro. The stoichiometry of variant nucleosomes in vivo may depend on several factors such as local concentrations but this brings forth yet another potential level of structural and functional heterogeneity.