Structural basis of instability of the nucleosome containing a testis-specific histone variant,human H3T

A histone H3 variant, H3T, is highly expressed in the testis, suggesting that it may play an important role in the chromatin reorganization required for meiosis and/or spermatogenesis. In the present study, we found that the nucleosome containing human H3T is significantly unstable both in vitro and in vivo, as compared to the conventional nucleosome containing H3.1. The crystal structure of the H3T nucleosome revealed structural differences in the H3T regions on both ends of the central α2 helix, as compared to those of H3.1. The H3T-specific residues (Met71 and Val111) are the source of the structural differences observed between H3T and H3.1. A mutational analysis revealed that these residues are responsible for the reduced stability of the H3T-containing nucleosome. These physical and structural properties of the H3T-containing nucleosome may provide the basis of chromatin reorganization during spermatogenesis.During spermatogenesis, dramatic chromatin reorganization occurs, and most histones are eventually replaced by protamines (1). Several histone variants are highly expressed in the testis and are considered to be incorporated into the chromatin in the early stage of spermatogenesis.In humans, about 4% of the haploid genome in the sperm is reportedly retained in nucleosomes, some containing the testis-specific histone H2B, hTSH2B/TH2B (2). Interestingly, the nucleosomes retained in the sperm are significantly enriched in loci that contain developmentally important genes. In addition, histone modifications, such as acetylation and methylation, are likely to occur after the incorporation of the histone variants during spermatogenesis (1). These observations suggest that nucleosomes containing testis-specific histone variants, with or without chemical modifications, may function as epigenetic markers in the sperm chromatin.H3T is a variant of histone H3 that is robustly expressed in the human testis (3–5). We previously reported that H3T, like the conventional H3.1, can be assembled into nucleosomes with H2A, H2B, and H4 (H3T nucleosome) (6). A histone chaperone, Nap2, with 3-fold higher expression in the testis than in other somatic tissues (7), was found to be a more efficient chaperone for H3T nucleosome assembly than the ubiquitously expressed histone chaperone, Nap1 (6). Therefore, H3T may be assembled into the chromatin by a specific chaperone-mediated pathway in the testis. Comprehensive proteome analyses of nuclear extracts from HeLa cells suggested that H3T also exists in somatic cells (8, 9). However, the nucleosomes containing H3T probably comprise only a small proportion of the bulk chromatin in somatic cells, because the amount of H3T in HeLa cells is extremely low. Therefore, H3T may have a limited function in somatic cells that is currently unknown.In the present study, we found that the H3T nucleosome is significantly unstable, as compared to the conventional H3.1 nucleosome, both in vitro and in vivo. The crystal structure of the H3T nucleosome was determined at 2.7 Å resolution, revealing that, although the overall structure was similar to that of the conventional H3.1 nucleosome, structural differences were observed at both ends of the central α2 helix of H3T and H3.1. The unique physical and structural characteristics of the H3T nucleosome were attributed to the Val111 and Met71 residues that are specific to H3T.