The role of H2B histones from the sea urchin sperm in the formation of supranucleosome structures

The structural role of histone H2B from sea urchin sperm (H2Bsp) has been examined in experiments on reconstitution of chromatin from DNA and core histones taken in three variants: (1) four core histones from sea urchin sperm; (2) four core histones from calf thymus; (3) (H3, H4, H2A) from calf thymus and H2Bsp. It is shown that H2Bsp when present in reconstituted chromatin induces its aggregation. Fidelity of the reconstitution of nucleosomes has been tested using DNase I probe, one- and two-dimensional electrophoresis and electron microscopy. The reconstitutes that contain H2Bsp appear under electron microscope mainly as regular closely spaced large granules, about 450 A in diameter, which are very similar to the granules found in "native" sea urchin sperm chromatin. The reconstitutes formed by four core histones from calf thymus appear as randomly arranged particles, about 100 A in diameter. We conclude that histone H2Bsp participates in interactions between nucleosomes and is involved in the formation of the condensed supranucleosomal structure in sea urchin sperm chromatin.     

Phosphorylation protects sperm-specific histones H1 and H2B from proteolysis after fertilization

At intermediate stages of male pronucleus formation, sperm-derived chromatin is composed of hybrid nucleoprotein particles formed by sperm H1 (SpH1), dimers of sperm H2A-H2B (SpH2A-SpH2B), and a subset of maternal cleavage stage (CS) histone variants. At this stage in vivo, the CS histone variants are poly(ADP-ribosylated), while SpH2B and SpH1 are phosphorylated. We have postulated previously that the final steps of sperm chromatin remodeling involve a cysteine-protease (SpH-protease) that degrades sperm histones in a specific manner, leaving the maternal CS histone variants unaffected. More recently we have reported that the protection of CS histones from degradation is determined by the poly(ADP-ribose) moiety of these proteins. Because of the selectivity displayed by the SpH-protease, the coexistence of a subset of SpH together with CS histone variants at intermediate stages of male pronucleus remodeling remains intriguing. Consequently, we have investigated the phosphorylation state of SpH1 and SpH2B in relation to the possible protection of these proteins from proteolytic degradation. Histones H1 and H2B were purified from sperm, phosphorylated in vitro using the recombinant alpha-subunit of casein kinase 2, and then used as substrates in the standard assay of the SpH-protease. The phosphorylated forms of SpH1 and SpH2B were found to remain unaltered, while the nonphosphorylated forms were degraded. On the basis of this result, we postulate a novel role for the phosphorylation of SpH1 and SpH2B that occurs in vivo after fertilization, namely to protect these histones against degradation at intermediate stages of male chromatin remodeling.     

Phosphorylation protects sperm‐specific histones H1 and H2B from proteolysis after fertilization

At intermediate stages of male pronucleus formation, sperm‐derived chromatin is composed of hybrid nucleoprotein particles formed by sperm H1 (SpH1), dimers of sperm H2A‐H2B (SpH2A‐SpH2B), and a subset of maternal cleavage stage (CS) histone variants. At this stage in vivo, the CS histone variants are poly(ADP‐ribosylated), while SpH2B and SpH1 are phosphorylated. We have postulated previously that the final steps of sperm chromatin remodeling involve a cysteine‐protease (SpH‐protease) that degrades sperm histones in a specific manner, leaving the maternal CS histone variants unaffected. More recently we have reported that the protection of CS histones from degradation is determined by the poly(ADP‐ribose) moiety of these proteins. Because of the selectivity displayed by the SpH‐protease, the coexistence of a subset of SpH together with CS histone variants at intermediate stages of male pronucleus remodeling remains intriguing. Consequently, we have investigated the phosphorylation state of SpH1 and SpH2B in relation to the possible protection of these proteins from proteolytic degradation. Histones H1 and H2B were purified from sperm, phosphorylated in vitro using the recombinant α‐subunit of casein kinase 2, and then used as substrates in the standard assay of the SpH‐protease. The phosphorylated forms of SpH1 and SpH2B were found to remain unaltered, while the nonphosphorylated forms were degraded. On the basis of this result, we postulate a novel role for the phosphorylation of SpH1 and SpH2B that occurs in vivo after fertilization, namely to protect these histones against degradation at intermediate stages of male chromatin remodeling. J. Cell. Biochem. 76:173–180, 1999. © 1999 Wiley‐Liss, Inc.     

The histones of the sperm of Spisula solidissima include a novel, cysteine-containing H-1 histone

The histones remaining at the end of the spermiogenic differentiation, which are found associated with a highly basic protamine-like component [Ausio, J. and K.E. Van Holde (1987) Eur. J. Biochem. 165, 363-371] in the mature sperm of Spisula solidissima, have been isolated and characterized for the first time. All four core histones H2A, H2B, H3, H4, and the lysine-rich histone H1 are present. The core histones are found in equal stoichiometric amounts. As has been observed in other bivalve molluscs, the amino acid compositions of the core histones of S. solidissima sperm are very close to those of their counterparts in the calf thymus somatic histones. The spermatic histone H1 exhibits an amino acid composition and structural features similar to other histones of the histone H1 family. Yet this latter histone seems to be sperm-specific, and it contains at least two cysteine residues per molecule, which makes it unique in its class.     

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.     

Degradation of sperm histones in vitro by cytoplasm of the sea urchin egg

Sperm-specific histone variants in the sea urchin Paracentrotus lividus are replaced early after fertilization with a specific embryonic set of histone variants. A possible in vitro model for the involvement of a degradation mechanism in the replacement of sperm-specific histones is presented. Soluble sperm histones are shown to be degraded quickly by egg cytoplasm. The proteolytic activity is maximal at pH 3.0; H1 and H2A histones are the most sensitive while H3 and H4 are the most resistant. H2B histones have an intermediate sensitivity. Histone degradation by egg cytoplasm or by purified fractions of it can be inhibited by chymostatin and leupeptin and, to a lesser degree, by pepstatin.     

Linker histones play a role in male meiosis and the development of pollen grains in tobacco

To examine the function of linker histone variants, we produced transgenic tobacco plants in which major somatic histone variants H1A and H1B were present at approximately 25% of their usual amounts in tobacco chromatin. The decrease in these major variants was accompanied by a compensatory increase in the four minor variants, namely, H1C to H1F. These minor variants are smaller and less highly charged than the major variants. This change offered a unique opportunity to examine the consequences to a plant of major remodeling of its chromatin set of linker histones. Plants with markedly altered proportions of H1 variants retained normal nucleosome spacing, but their chromosomes were less tightly packed than those of control plants. The transgenic plants grew normally but showed characteristic aberrations in flower development and were almost completely male sterile. These features correlated with changes in the temporal but not the spatial pattern of expression of developmental genes that could be linked to the abnormal flower phenotypes. Preceding these changes in flower morphology were strong aberrations in male gametogenesis. The earliest symptoms may have resulted from disturbances in correct pairing or segregation of homologous chromosomes during meiosis. No aberrations were observed during mitosis. We conclude that in plants, the physiological stoichiometry and distribution of linker histone variants are crucial for directing male meiosis and the subsequent development of functional pollen grains.     

Interaction of sperm histone variants and linker DNA during spermiogenesis in the sea urchin

Several physical properties of sea urchin spermatid chromatin, which contains phosphorylated Sp H1 and Sp H2B histone variants, and mature sperm chromatin, in which these histones are dephosphorylated, were compared. Density, thermal stability, average nucleosomal repeat length, and resistance to micrococcal nuclease digestion are all increased in mature sperm relative to spermatid chromatin. Since the chromatins are identical in histone variant subtypes, the altered physical properties are not a consequence of changes in histone primary structure during spermiogenesis. The data are interpreted to mean that dephosphorylation of the N-terminal regions of Sp H1 and Sp H2B in late spermatid nuclei permits strong ionic binding of these highly basic regions to the extended linker, stabilizing the highly condensed structure of sperm chromatin.     

Analysis of histones in Xenopus laevis. I. A distinct index of enriched variants and modifications exists in each cell type and is remodeled during developmental transitions

Histone proteins contain epigenetic information that is encoded both in the relative abundance of core histones and variants and particularly in the post-translational modification of these proteins. We determined the presence of such variants and covalent modifications in seven tissue types of the anuran Xenopus laevis, including oocyte, egg, sperm, early embryo equivalent (pronuclei incubated in egg extract), S3 neurula cells, A6 kidney cells, and erythrocytes. We first developed a new robust method for isolating the stored, predeposition histones from oocytes and eggs via chromatography on heparin-Sepharose, whereas we isolated chromatinized histones via conventional acid extraction. We identified two previously unknown H1 isoforms (H1fx and H1B.Sp) present on sperm chromatin. We immunoblotted this global collection of histones with many specific post-translational modification antibodies, including antibodies against methylated histone H3 on Lys(4), Lys(9), Lys(27), Lys(79), Arg(2), Arg(17), and Arg(26); methylated histone H4 on Lys(20); methylated H2A and H4 on Arg(3); acetylated H4 on Lys(5), Lys(8), Lys(12), and Lys(16) and H3 on Lys(9) and Lys(14); and phosphorylated H3 on Ser(10) and H2A/H4 on Ser(1). Furthermore, we subjected a subset of these histones to two-dimensional gel analysis and subsequent immunoblotting and mass spectrometry to determine the global remodeling of histone modifications that occurs as development proceeds. Overall, our observations suggest that each metazoan cell type may have a unique histone modification signature correlated with its differentiation status.     

Remodeling of sperm chromatin after fertilization involves nucleosomes formed by sperm histones H2A and H2B and two CS histone variants

The composition of nucleosomes at an intermediate stage of male pronucleus formation was determined in sea urchins. Nucleosomes were isolated from zygotes harvested 10 min post-insemination, whole nucleoprotein particles were obtained from nucleus by nuclease digestion, and nucleosomes were subsequently purified by a sucrose gradient fractionation. The nucleosomes derived from male pronucleus were separated from those derived from female pronucleus by immunoadsorption to antibodies against sperm specific histones (anti-SpH) covalently bound to Sepharose 4B (anti-SpH-Sepharose). The immunoadsorbed nucleosomes were eluted, and the histones were analyzed by Western blots. Sperm histones (SpH) or alternatively, the histones from unfertilized eggs (CS histone variants), were identified with antibodies directed against each set of histones. It was found that these nucleosomes are organized by a core formed by sperm histones H2A and H2B combined with two major CS histone variants. Such a hybrid histone core interacts with DNA fragments of approximately 100 bp. It was also found that these atypical nucleosome cores are subsequently organized in a chromatin fiber that exhibits periodic nuclease hypersensitive sites determined by DNA fragments of 500 bp of DNA. It was found that these nucleoprotein particles were organized primarily by the hybrid nucleosomes described above. We postulate that this unique chromatin organization defines an intermediate stage of male chromatin remodeling after fertilization.     

Histones and nucleosomes in Cancer sperm (Decapod: Crustacea) previously described as lacking basic DNA-associated proteins: a new model of sperm chromatin

To date several studies have been carried out which indicate that DNA of crustacean sperm is neither bound nor organized by basic proteins and, contrary to the rest of spermatozoa, do not contain highly packaged chromatin. Since this is the only known case of this type among metazoan cells, we have re-examined the composition, and partially the structure, of the mature sperm chromatin of Cancer pagurus, which has previously been described as lacking basic DNA-associated proteins. The results we present here show that: (a) sperm DNA of C. pagurus is bound by histones forming nucleosomes of 170 base pairs, (b) the ratio [histones/DNA] in sperm of two Cancer species is 0.5 and 0.6 (w/w). This ratio is quite lower than the proportion [proteins/DNA] that we found in other sperm nuclei with histones or protamines, whose value is from 1.0 to 1.2 (w/w), (c) histone H4 is highly acetylated in mature sperm chromatin of C. pagurus. Other histones (H3 and H2B) are also acetylated, though the level is much lower than that of histone H4. The low ratio of histones to DNA, along with the high level of acetylation of these proteins, explains the non-compact, decondensed state of the peculiar chromatin in the sperm studied here. In the final section we offer an explanation for the necessity of such decondensed chromatin during gamete fertilization of this species.     

Structure and functions of linker histones

Linker histones such as variants H1, H5, and other similar proteins play an important role in regulation of chromatin structure and dynamics. However, interactions of linker histones with DNA and proteins, as well as specific functions of their different variants, are poorly studied. This is because they acquire tertiary structure only when interacting with a nucleosome, and because of limitations of currently available methods. However, deeper investigation of linker histones and their interactions with other proteins will address a number of important questions — from structure of compacted chromatin to regulation of early embryogenesis. In this review, structures of histone H1 variants and its interaction with chromatin DNA are considered. A possible functional significance of different H1 variants, a role of these proteins in maintaining interphase chromatin structure, and interactions of linker histones with other cellular proteins are also discussed.     

Changes in the histones of the sea urchin Strongylocentrotus purpuratus at fertilization

Both sperm and eggs of the sea urchin Strongylocentrotus purpuratus contain specific histones in place of some of the histones found during later development. Whether these specific histones are lost upon fertilization or are retained is not known. Therefore, we have examined the histones present in the zygote nucleus to determine the fate of the gamete histones. Nuclei of zygotes which have completed DNA replication in preparation for the first mitosis were isolated by sucrose density gradient centrifugation. Histones were extracted from the isolated nuclei, and were analyzed by acid-urea and SDS polyacrylamide gel electrophoreses, and by two-dimensional electrophoresis in which both gel electrophoresis systems were combined. Electrophoretic patterns of the zygote histones were compared with those of sperm, unfertilized eggs and embryos. The results show that the zygote histone pattern is identical with the unfertilized egg histone pattern. Neither the sperm histones H1, H2A, or H2B, nor the embryonic H1, H2A, or H2B, are present in the zygote pattern. The egg and the zygote do contain a unique H2A and H2B, but not an H1. After fertilization, sperm specific histones are not present on the DNA. Egg histones become associated with both the sperm DNA and the newly replicated DNA. The association of the embryonic histones with the DNA, therefore, occurs sometime later in development.     

Immunobiochemical evidence for the loss of sperm specific histones during male pronucleus formation in monospermic zygotes of sea urchins

To obtain information on the remodeling of sperm chromatin during male pronuclei formation, we have followed the sperm specific histones (SpH) that form the nucleosomal core by Western immunoblot analysis with polyclonal antibodies directed against the core SpH. The results obtained indicate that the complete set of SpH is absent from zygote chromatin at the beginning of the first S phase. The disappearance of SpH is not coincidental for the five histone classes: SpH4 and SpH3 are lost 5-15 min post insemination (p.i.), SpH2B and SpH2A disappear 20-40 min p.i., and SpH1 is progressively diminished up to 30 min p.i. This order of sperm chromatin remodeling is not affected by the inhibition of protein synthesis by emetine, indicating that the factor(s) responsible for SpH disappearance are present in unfertilized eggs. The lost SpH's are not replaced by newly synthesized CS variants, since the basic proteins synthesized de novo during male pronuclei formation are not incorporated into chromatin remaining in the cytoplasm. These newly synthesized proteins are different from the CS variants as judged by their electrophoretic migration.     

Preliminary study of sperm chromatin characteristics of the brachyuran crab Maja brachydactyla. Histones and nucleosome-like structures in decapod crustacean sperm nuclei previously described without SNBPs

An interesting characteristic of decapod crustacean sperm nuclei is that they do not contain highly packaged chromatin. In the present study we re-examine the presence of DNA-interacting proteins in sperm nuclei of the brachyuran Maja brachydactyla. Although previous reports have indicated that, unlike the majority of sperm cells, DNA of decapod sperm is not organized by basic proteins, in this work we show that: (1) histones are present in sperm of M. brachydactyla; (2) histones are associated with sperm DNA; (3) histone H3 appears in lower proportions than the other core histones, while histone H2B appears in higher proportions; and (4) histone H3 in sperm nuclei is acetylated. This work complements a previous study of sperm histones of Cancer pagurus and supports the suggestion that decapod crustacean sperm chromatin deserves further attention.