Characterization of nucleosomes consisting of the human testis/sperm-specific histone H2B variant (hTSH2B)

We have reported earlier the occurrence of a specific histone H2B variant in human testis and sperm. Here we have structurally characterized this protein, its association with the rest of the histone octamer, and its effects on the nucleosome structure. We show that a reconstituted octamer consisting of hTSH2B and a stoichiometric complement of histones H2A, H3, and H4 exhibits a lower stability compared to the reconstituted native counterpart consisting of H2B. In contrast, the hTSH2B containing octamers are able to form nucleosome core particles which are structurally and dynamically indistinguishable from those reconstituted with octamers consisting of only native histones. Furthermore, the presence of hTSH2B in the nucleosome does not affect its ability to bind to linker histones.     

H2A.Bbd: an X-chromosome-encoded histone involved in mammalian spermiogenesis

Despite the identification of H2A.Bbd as a new vertebrate-specific replacement histone variant several years ago, and despite the many in vitro structural characterizations using reconstituted chromatin complexes consisting of this variant, the existence of H2A.Bbd in the cell and its location has remained elusive. Here, we report that the native form of this variant is present in highly advanced spermiogenic fractions of mammalian testis at the time when histones are highly acetylated and being replaced by protamines. It is also present in the nucleosomal chromatin fraction of mature human sperm. The ectopically expressed non-tagged version of the protein is associated with micrococcal nuclease-refractory insoluble fractions of chromatin and in mouse (20T1/2) cell line, H2A.Bbd is enriched at the periphery of chromocenters. The exceedingly rapid evolution of this unique X-chromosome-linked histone variant is shared with other reproductive proteins including those associated with chromatin in the mature sperm (protamines) of many vertebrates. This common rate of evolution provides further support for the functional and structural involvement of this protein in male gametogenesis in mammals.     

Molecular cloning and differential expression of somatic and testis-specific H2B histone genes during rat spermatogenesis

We have cloned cDNA of a testis-specific histone, TH2B (a variant of H2B), and rat somatic H2B gene to investigate regulation of testis-specific histone genes during rat spermatogenesis. The amino acid sequences deduced from DNA sequences show extensive sequence divergence in the N-terminal third of the two histones. The rest is highly conserved. One cysteine residue was found in TH2B. No cysteine is present in somatic histones except in H3 histone. We investigated the expression of TH2B and H2B genes using the regions of sequence divergence as hybridization probes. The TH2B gene is expressed only in the testis, and the expression of this gene is detected 14 days after birth, reaching a maximum at Day 20. The level of H2B mRNA shows a reciprocal pattern. This contrasting pattern can be explained by the gradually changing proportion of spermatogonia and spermatocytes with testicular maturation. In situ cytohybridization studies show that H2B gene is expressed primarily in proliferating spermatogonia and preleptotene spermatocytes, whereas TH2B gene is expressed exclusively in pachytene spermatocytes which first appear in testis about 14 days after birth. H2B and TH2B genes appear to be ideal markers for the study of proliferation and differentiation events in spermatogenesis and their regulatory mechanisms.     

Human testis-specific histone TH2B: Fractionation and peptide mapping

The presence of the nonionic detergent Triton X-100 was shown to improve the resolution of the human TH2B on gel electrophoresis and on gel filtration. Total histones of human testis, including TH2B, were resolved by electrophoresis in 15% polyacrylamide gels containing 0.4% Triton X-100, 1.5 m urea, and 0.9 n acetic acid. Gel filtration on Bio-Gel P-200 in 0.4% Triton X-100, 5.0 m urea, and 0.01 n HCl permitted the purification of human TH2B from human testis and sperm in preparative amounts. The structure of human TH2B so prepared was compared to that of rat TH2B, human H2B, and rat H2B by tryptic peptide mapping. The results showed some similarities between all four proteins, but closer similarity was observed within the germ cell histone (TH2B) group and within the somatic histone (H2B) group than between histones of the same species. In addition, human TH2B and rat TH2B each contained one unique peptide absent from other histones.     

A human histone H2B.1 variant gene, located on chromosome 1, utilizes alternative 3' end processing.

A variant human H2B histone gene (GL105), previously shown to encode a 2300 nt replication independent mRNA, has been cloned. We demonstrate this gene expresses alternative mRNAs regulated differentially during the HeLa S3 cell cycle. The H2B-Gl105 gene encodes both a 500 nt cell cycle dependent mRNA and a 2300 nt constitutively expressed mRNA. The 3' end of the cell cycle regulated mRNA terminates immediately following the region of hyphenated dyad symmetry typical of most histone mRNAs, whereas the constitutively expressed mRNA has a 1798 nt non-translated trailer that contains the same region of hyphenated dyad symmetry but is polyadenylated. The cap site for the H2B-GL105 mRNAs is located 42 nt upstream of the protein coding region. The H2B-GL105 histone gene was localized to chromosome region 1q21-1q23 by chromosomal in situ hybridization and by analysis of rodent-human somatic cell hybrids using an H2B-GL105 specific probe. The H2B-GL105 gene is paired with a functional H2A histone gene and this H2A/H2B gene pair is separated by a bidirectionally transcribed intergenic promoter region containing consensus TATA and CCAAT boxes and an OTF-1 element. These results demonstrate that cell cycle regulated and constitutively expressed histone mRNAs can be encoded by the same gene, and indicate that alternative 3' end processing may be an important mechanism for regulation of histone mRNA. Such control further increases the versatility by which cells can modulate the synthesis of replication-dependent as well as variant histone proteins during the cell cycle and at the onset of differentiation.     

Acetylation of rat testis histones H2B and TH2B

The in vivo acetylation of rat testis histones H3 and H4 has been demonstrated in previous studies. In this study, analysis of purified histone fractions revealed the in vivo acetylation of histone H2B, the testis histone variant designated TH2B, and two or more of the histone H2A variants. These findings are quite significant, because it is possible that all of the core histones are acetylated in elongating spermatids at the time of removal of the entire histone complement for replacement by basic spermatidal transition proteins (S.R. Grimes and N. Henderson, 1983, Arch. Biochem. Biophys. 221, 108-116).     

Expression and purification of recombinant human histones

Nucleosomes reconstituted from bacterially expressed histones are useful for functional and structural analyses of histone variants, histone mutants, and histone post-translational modifications. In the present study, we developed a new method for the expression and purification of recombinant human histones. The human histone H2A, H2B, and H3 genes were expressed well in Escherichia coli cells, but the human histone H4 gene was poorly expressed. Therefore, we designed a new histone H4 gene with codons optimized for the E. coli expression system and constructed the H4 gene by chemically synthesized oligodeoxyribonucleotides. The recombinant human histones were expressed as hexahistidine-tagged proteins and were purified by one-step chromatography with nickel-nitrilotriacetic acid agarose in the presence of 6 M urea. The H2A/H2B dimer and the H3/H4 tetramer were refolded by dialysis against buffer without urea, and the hexahistidine-tags of the histones in the H2A/H2B dimer and the H3/H4 tetramer were removed by thrombin protease digestion. The H2A/H2B dimer and the H3/H4 tetramer obtained by this method were confirmed to be proficient in nucleosome formation by the salt dialysis method. The human CENP-A gene, the centromere-specific histone H3 variant, contains 28 minor codons for E. coli. A new CENP-A gene optimized for the E. coli expression system was also constructed, and we found that the purified recombinant CENP-A protein formed a nucleosome-like structure with histones H2A, H2B, and H4.     

Basic nuclear proteins in testicular cells and ejaculated spermatozoa in man

Human testis was shown to contain a specific histone, TH2B, having the same electrophoretic mobility as rat TH2B. Testicular and ejaculated human sperm still possessed histones at 50% and 15% of the total basic nuclear proteins, respectively. Comparison of the electrophoretic patterns of histones from human testis, testicular sperm and ejaculated sperm implied that the histones may be removed in the order H2A and H1 before H3, H4 and H2B before TH2B. TH2B which is the major histone fraction in ejaculated sperm has no longer a strong affinity to DNA. TH2B in sperm nuclei could be separated from other basic nuclear proteins by Bio-Gel P-10 column chromatography and its amino acid composition is similar to that of rat TH2B, although no cysteine residue was found.     

A histone H2B variant from the embryo of the sea urchin Parenchinus angulosus

A variant of histone H2B has been isolated from sea urchin embryo (Parenchinus angulosus). Out of the 53 amino acids positioned in the three CNBr-peptides only 26 residues are identical to those in the corresponding positions of calf thymus histone H2B. A similar degree of homology exists between the embryonic variant and the previously characterized variants from sperm cells of the same organism.     

Galactosyl receptor in human testis and sperm is antigenically related to the minor C-type (Ca2+-dependent) lectin variant of human and rat liver

Galactosyl receptor, a cell surface Ca²⁺-dependent lectin with binding affinity for galactose, was evaluated by immunoblotting, immunoprecipitation, Northern blotting, and immunocytochemistry in human liver, testis, and sperm. Polyclonal antisera raised against the minor asialoglycoprotein receptor variant of rat hepatocytes (designated rat hepatic lectin-2/3, RHL-2/3), and its human liver-equivalent (designated H2), recognize native galactosyl receptor in the testis and sperm in immunoblotting, immunoprecipitation, and immunocytochemical experiments. An equivalent to the major hepatocyte asialoglycoprotein receptor variant (rat RHL-1 and human H1) was not detected. Human testis and sperm galactosyl receptor was resolved, after immunoprecipitation and immunoblotting, as a single protein component of molecular mass 50 kD. The single protein component in human testis and sperm contrasted with the doublet nature of rat testis and sperm galactosyl receptor, consisting of two components of molecular masses of 54 and 49 kD. Northern blotting experiments using radiolabeled H1 and H2 cDNA probes confirmed the presence of H2 mRNA and the lack of H1 mRNA in the human testis. Immunocytochemical studies detected specific antigenic sites on the entire surfaces of spermatogenic cells. However, immunoreactivity in epididymal and ejaculated sperm was confined to head surfaces overlying the acrosome. Results from these studies, and from previous studies in the rat, suggest that the testis/sperm galactosyl receptor is a C-type Ca²⁺-dependent lectin with possible roles in cell-cell interaction during spermatogenesis and sperm-zona pellucida binding at fertilization. © 1995 Wiley-Liss, Inc.     

Human sperm telomere-binding complex involves histone H2B and secures telomere membrane attachment

Telomeres are unique chromatin domains located at the ends of eukaryotic chromosomes. Telomere functions in somatic cells involve complexes between telomere proteins and TTAGGG DNA repeats. During the differentiation of germ-line cells, telomeres undergo significant reorganization most likely required for additional specific functions in meiosis and fertilization. A telomere-binding protein complex from human sperm (hSTBP) has been isolated by detergent treatment and was partially purified. hSTBP specifically binds double-stranded telomeric DNA and does not contain known somatic telomere proteins TRF1, TRF2, and Ku. Surprisingly, the essential component of this complex has been identified as a specific variant of histone H2B. Indirect immunofluorescence shows punctate localization of H2B in sperm nuclei, which in part coincides with telomeric DNA localization established by fluorescent in situ hybridization. Anti-H2B antibodies block interactions of hSTBP with telomere DNA, and spH2B forms specific complex with this DNA in vitro, indicating that this protein plays a role in telomere DNA recognition. We propose that hSTBP participates in the membrane attachment of telomeres that may be important for ordered chromosome withdrawal after fertilization.     

Structural change of a particular H2B histone gene possibly results in differences in its transcriptional regulation in different chicken tissues.

The chicken H2B histone gene family consists of eight highly homologous members (H2B-I to H2B-VIII) belonging to two major histone gene clusters. Seven of these genes have been sequenced and shown to encode three different H2B protein variants. Northern analysis with a probe, which mainly consists of the 5'-flanking region containing the sequence for the mRNA leader of H2B-V encoding a particular H2B protein variant, revealed that the mRNA level transcribed from this particular gene was higher in the kidney than in the oviduct and lung. To elucidate whether the structure of the H2B gene differs in the three different tissues, we analyzed DNAs from the oviduct, lung, and kidney. On Southern analysis, various H2B gene-specific probes hybridized with two particular H2B genes (H2B-IV and H2B-V), which are located in close proximity within a 12 kb EcoRI fragment, from the oviduct and lung with an intensity of about one quarter of that from the kidney. These findings suggest that some difference of DNA structure of the H2B-V gene may result in its relatively higher expression in the kidney.     

H2B ubiquitylation: the end is in sight

Historically, the first eukaryotic protein found to be modified by ubiquitin was H2A, originally isolated from HeLa cells in 1975 by Harrison Busch and coworkers as a histone-like, nonhistone chromosomal protein called A24. Ubiquitylated histones have subsequently been found in many eukaryotic species, and to date, the core histones H2A, H2B, H3, the linker histone H1, and the histone variant H2A.Z are known to carry this modification. Although first on the scene, it was only recently that studies on histone ubiquitylation have enjoyed a renaissance. Part of the reason for the relatively slow pace of research on this fascinating histone modification was the absence of a good genetic system with which to study its cellular roles. This changed in 2000, when histone H2B was found to be ubiquitylated in the budding yeast S. cerevisiae, an organism with a low histone gene copy number and highly tractable genetics. Another factor was the almost exclusive focus of research on the role of polyubiquitylation in protein turnover. Because histones are generally monoubiquitylated, a form of the modification that is not associated with protein degradation, the significance of this minimalist ubiquitin conjugation was not heavily pursued. But perhaps the key reason for the renewed interest in histone ubiquitylation was the unexpected discovery of the past year that ubiquitylated H2B plays an important role in the trans-histone methylation of histone H3, a modification with close ties to the regulation of gene expression. This review will highlight some of the recent findings on the regulation and cellular roles of H2B ubiquitylation in yeast.