ABSTRACT
Histone replacement by transition proteins (TPs) and protamines (Prms) constitutes an essential step for the successful production of functional male gametes, yet nothing is known on the underlying functional interplay between histones, TPs, and Prms. Here, by studying spermatogenesis in the absence of a spermatid-specific histone variant, H2A.L.2, we discover a fundamental mechanism involved in the transformation of nucleosomes into nucleoprotamines. H2A.L.2 is synthesized at the same time as TPs and enables their loading onto the nucleosomes. TPs do not displace histones but rather drive the recruitment and processing of Prms, which are themselves responsible for histone eviction. Altogether, the incorporation of H2A.L.2 initiates and orchestrates a series of successive transitional states that ultimately shift to the fully compacted genome of the mature spermatozoa. Hence, the current view of histone-to-nucleoprotamine transition should be revisited and include an additional step with H2A.L.2 assembly prior to the action of TPs and Prms.
Subject(s)
Chromatin Assembly and Disassembly , Chromatin/metabolism , Histones/metabolism , Nucleosomes/metabolism , Protamines/metabolism , Spermatogenesis , Spermatozoa/metabolism , Animals , COS Cells , Chlorocebus aethiops , Chromatin/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Computational Biology , Databases, Genetic , Fertility , Gene Expression Regulation, Developmental , Genetic Predisposition to Disease , Genome , Histones/deficiency , Histones/genetics , Infertility, Male/genetics , Infertility, Male/metabolism , Infertility, Male/pathology , Infertility, Male/physiopathology , Male , Mice, 129 Strain , Mice, Knockout , Nucleosomes/genetics , Phenotype , Spermatogenesis/genetics , Spermatozoa/pathology , TransfectionABSTRACT
Cellular differentiation is associated with dynamic chromatin remodeling in establishing a cell-type-specific epigenomic landscape. Here, we find that mouse testis-specific and replication-dependent histone H3 variant H3t is essential for very early stages of spermatogenesis. H3t gene deficiency leads to azoospermia because of the loss of haploid germ cells. When differentiating spermatogonia emerge in normal spermatogenesis, H3t appears and replaces the canonical H3 proteins. Structural and biochemical analyses reveal that H3t-containing nucleosomes are more flexible than the canonical nucleosomes. Thus, by incorporating H3t into the genome during spermatogonial differentiation, male germ cells are able to enter meiosis and beyond.
Subject(s)
Histones/genetics , Spermatogonia/metabolism , Testis/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Animals , Azoospermia/etiology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Differentiation , Cells, Cultured , Chromatin/chemistry , Chromatin/metabolism , Germ Cells/cytology , Germ Cells/metabolism , Haploidy , Histones/chemistry , Histones/deficiency , Male , Meiosis , Mice , Mice, Inbred C57BL , Mice, Knockout , Nucleosomes/chemistry , Nucleosomes/metabolism , Spermatogenesis , Testis/pathologyABSTRACT
BACKGROUND: Human histone H3.5 is a non-allelic H3 variant evolutionally derived from H3.3. The H3.5 mRNA is highly expressed in human testis. However, the function of H3.5 has remained poorly understood. RESULTS: We found that the H3.5 nucleosome is less stable than the H3.3 nucleosome. The crystal structure of the H3.5 nucleosome showed that the H3.5-specific Leu103 residue, which corresponds to the H3.3 Phe104 residue, reduces the hydrophobic interaction with histone H4. Mutational analyses revealed that the H3.5-specific Leu103 residue is responsible for the instability of the H3.5 nucleosome, both in vitro and in living cells. The H3.5 protein was present in human seminiferous tubules, but little to none was found in mature sperm. A chromatin immunoprecipitation coupled with sequencing analysis revealed that H3.5 accumulated around transcription start sites (TSSs) in testicular cells. CONCLUSIONS: We performed comprehensive studies of H3.5, and found the instability of the H3.5 nucleosome and the accumulation of H3.5 protein around TSSs in human testis. The unstable H3.5 nucleosome may function in the chromatin dynamics around the TSSs, during spermatogenesis.
ABSTRACT
The human histone H2B variant TSH2B is highly expressed in testis and may function in the chromatin transition during spermatogenesis. In the present study, the crystal structure of the human testis-specific nucleosome containing TSH2B was determined at 2.8â Å resolution. A local structural difference between TSH2B and canonical H2B in nucleosomes was detected around the TSH2B-specific amino-acid residue Ser85. The TSH2B Ser85 residue does not interact with H4 in the nucleosome, but in the canonical nucleosome the H2B Asn84 residue (corresponding to the TSH2B Ser85 residue) forms water-mediated hydrogen bonds with the H4 Arg78 residue. In contrast, the other TSH2B-specific amino-acid residues did not induce any significant local structural changes in the TSH2B nucleosome. These findings may provide important information for understanding how testis-specific histone variants form nucleosomes during spermatogenesis.
