Human sperm, unlike the sperm of other mammals, contain replacement histones with unknown biological functions. Here, we report the identification of the novel human gene coding for a testis/sperm-specific histone H2B (hTSH2B). This variant histone is 85% homologous to somatic H2B and has over 93% homology with the testis H2B of rodents. Using genomic PCR, two genetic alleles of hTSH2B were found in the human population. The hTSH2B gene is transcribed exclusively in testis, and the corresponding protein is also present in mature sperm. We expressed recombinant hTSH2B and identified this protein with a particular H2B subtype expressed in vivo. The subnuclear distribution of H2B variants in sperm was determined using biochemical fractionation and immunoblotting. The H2B variant associated with telomere-binding activity (15) was solubilized by Triton X-100 or micrococcal nuclease extraction, whereas hTSH2B was relatively tightly bound in nuclei. Immunofluorescence showed that hTSH2B was concentrated in spots located at the basal nuclear area of a subpopulation (20% of cells) of mature sperm. This fact may be of particular importance, because the hTSH2B "positive" and "negative" sperm cells may undergo significantly different decondensation processes following fertilization.During mammalian spermatogenesis, chromatin undergoes stage-specific structural reorganization. Testis-specific variants of histones, transitional proteins, and finally protamines replace somatic histones as the DNA condenses (1, 2). The complement of spermatogenic histones changes during differentiation, and this process has been described in detail for rodents (1). Whereas some testis histones (TH) 1 appear in the early stages of spermatogenesis for instance in spermatogonia (3) and some appear at late stages (e.g. in spermatids (4, 5)), the majority of TH are synthesized and incorporated into chromatin during meiosis (6). Rodent testis histones are replacement subtypes that differ in primary structure from the major somatic variants (3-5, 7-9). Although it is generally assumed that histone variants of germ line cells contribute to the restructuring of chromatin during spermatogenesis, their specific biological functions remain to be established.In contrast to other mammals, mature human sperm retains a set of core histones representing 10 -15% of basic proteins (10 -12). Of these, histone H2B fraction (hSH2B) is the most abundant (10, 13). A complex composition of the hSH2B has been indicated (12). Although 17 replication-dependent H2B genes have been identified so far in the human genome (14), this complement contains neither testis-nor sperm-specific genes. We were particularly interested in the characterization of sperm H2B because of our recent finding that a H2B-related protein is an essential part of the telomere-binding complex in human sperm (15).In this paper, the hSH2B group has been characterized using a combination of biochemical and immunochemical techniques. Further, a gene for a novel human histone H2B variant belonging to thi...

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Background: DNA strand exchange proteins Dmc1 and Rad51 and translocases Tid1 and Rad54 function in DNA break repair during meiosis.Results: We biochemically demonstrate that Dmc1 and Rad51 are specifically stimulated by Tid1 and Rad54, respectively. Conclusion: Dmc1-Tid1 and Rad51-Rad54 represent functional pairs for DNA pairing and joint molecule formation. Significance: The separate and independent functioning of these proteins offers insight into DNA pairing in meiosis.

Histone acetylation, methylation, and phosphorylation occur predominantly in the unstructured N-terminal domains or histone "tails". These modifications and others comprise a "histone code" that directly facilitates or antagonizes association of regulatory proteins with nucleosomes to mediate changes in chromatin structure and activity. Methylation of histone H3 outside of the tail region at lysine 79 has been reported for a variety of species ranging from yeast to humans and in some gene-specific cases appears to be associated with active chromatin and transcription. Whether methylation of lysine 79 is associated with other post-translational modifications of the H3 tail is unknown. Using mass spectrometric relative quantitation, a mass spectrometric "Western blot", we compare methylation at lysines 4, 9, and 79 with acetylation of human histone H3. We find that the total levels of lysine 4 and 79 methylation (combined mono-, di-, and trimethylation) in the H3 population increase with the degree of H3 tail acetylation. The total amount of lysine 4 methylation increases progressively from less than 10% in the nonacetylated H3 to greater than 90% in the penta-acetylated H3. In addition, significant levels of lysine 4 trimethylation also occur in combination with the penta-acetylated H3 species. In contrast, the level of H3 lysine 9 trimethylation is greatest for the monoacetylated species while H3 lysine 9 acetylation occurs predominantly in hyperacetylated (tetra- and penta-acetylated) H3 isoforms. Together, these results indicate that methylation of lysine 4 and 79 as well as the switch from lysine 9 methylation to acetylation are coordinated synchronously with H3 hyperacetylation as marks of active chromatin.

The acetylation isoforms of histone H4 from butyratetreated HeLa cells were separated by C 4 reverse-phase high pressure liquid chromatography and by polyacrylamide gel electrophoresis. Histone H4 bands were excised and digested in-gel with the endoprotease trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to characterize the level of acetylation, and nanoelectrospray tandem mass spectrometric analysis of the acetylated peptides was used to determine the exact sites of acetylation. Although there are 15 acetylation sites possible, only four acetylated peptide sequences were actually observed. The tetra-acetylated form is modified at lysines 5, 8, 12, and 16, the tri-acetylated form is modified at lysines 8, 12, and 16, and the di-acetylated form is modified at lysines 12 and 16. The only significant amount of the mono-acetylated form was found at position 16. These results are consistent with the hypothesis of a "zip" model whereby acetylation of histone H4 proceeds in the direction of from Lys-16 to Lys-5, and deacetylation proceeds in the reverse direction. Histone acetylation and deacetylation are coordinated processes leading to a non-random distribution of isoforms. Our results also revealed that lysine 20 is dimethylated in all modified isoforms, as well as the nonacetylated isoform of H4.