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.     

Electrophoretic analysis of the stored histone pool in unfertilized sea urchin eggs: quantification and identification by antibody binding

A maternal store of histones in unfertilized sea urchin eggs is demonstrated by two independent criteria. Stored histones are identified by their ability to assemble into chromatin of male pronuclei of fertilized sea urchin eggs in the absence of protein synthesis, suggesting a minimum of at least 25 haploid equivalents for each histone present and functional in the unfertilized egg. In addition, electrophoretic analysis of proteins from acid extracts of unfertilized whole eggs and enucleated merogons reveals protein spots comigrating with cleavage stage histone standards, though not with other histone variants found in later sea urchin development or in sperm. Quantification of the amount of protein per histone spot yields an estimate of several hundred haploid DNA equivalents per egg of stored histone. The identity of some of the putative histones was verified by a highly sensitive immunological technique, involving electrophoretic transfer of proteins from the two-dimensional polyacrylamide gels to nitrocellulose filters. Proteins in amounts less than 2 x 10(-4) micrograms can be detected by this method.     

Evidence for the nuclear import of histones H3.1 and H4 as monomers

Newly synthesised histones are thought to dimerise in the cytosol and undergo nuclear import in complex with histone chaperones. Here, we provide evidence that human H3.1 and H4 are imported into the nucleus as monomers. Using a tether‐and‐release system to study the import dynamics of newly synthesised histones, we find that cytosolic H3.1 and H4 can be maintained as stable monomeric units. Cytosolically tethered histones are bound to importin‐alpha proteins (predominantly IPO4), but not to histone‐specific chaperones NASP, ASF1a, RbAp46 (RBBP7) or HAT1, which reside in the nucleus in interphase cells. Release of monomeric histones from their cytosolic tether results in rapid nuclear translocation, IPO4 dissociation and incorporation into chromatin at sites of replication. Quantitative analysis of histones bound to individual chaperones reveals an excess of H3 specifically associated with sNASP, suggesting that NASP maintains a soluble, monomeric pool of H3 within the nucleus and may act as a nuclear receptor for newly imported histone. In summary, we propose that histones H3 and H4 are rapidly imported as monomeric units, forming heterodimers in the nucleus rather than the cytosol.     

Deposition of newly synthesized histones: misinterpretations due to cross-linking density-labeled proteins with Lomant's reagent

We have reinvestigated studies using Lomant's reagent to cross-link newly synthesized density-labeled histones into octameric complexes to determine the nature of histone deposition. The analysis has additionally included procedures for reversal of the cross-link in order to analyze the individual histones in these complexes. These studies indicate that density-labeled, newly synthesized histones form hybrid octameric structures composed of both new and old histones. These studies also suggest that previous interpretations by other investigators for the production of homogeneous complexes (100% dense octamer containing 100% new histones) are misinterpretations due to the presence of non-histone protein that contaminates the preparation, even under conditions where much of this non-histone protein is removed by use of ion-exchange resins. These non-histone proteins can be fractionated on density gradients as un-cross-linked proteins with molecular weights that mimic those of cross-linked histone complexes.     

Histone synthesis in early amphibian development: histone and DNA syntheses are not co-ordinated

The synthesis of basic proteins has been studied in the oocytes, eggs and embryos of the South African clawed frog, Xenopus laevis. A group of newly synthesized proteins has been identified as histones by the following criteria: solubility properties; incorporation of [³H]lysine and [³H]arginine in the correct proportions, but lack of incorporation of [³H]tryptophan; co-cleotrophoresis with marker histones in various types of polyacrylamide gels, including a type run in two dimensions; peptide analysis of the arginine-rich fraction, F2A1. The four main histone fractions other than F1 were found to be synthesized at all stages of development. F1 histone synthesis was first detected at the late blastula stage.Rates of histone synthesis were estimated for the different stages of development and it was concluded that histone synthesis was not co-ordinated with DNA synthesis either temporally or quantitatively. Histone synthesis was unusual in the following major respects: histones were synthesized in oocytes, and yet in these cells DNA replication had not occurred for several months; histones were synthesized in activated or fertilized eggs at a rate far in excess (about 500 times) of the immediate requirements. We suggest that in order to provide enough histones for the late blastula embryo a store of histone is accumulated during the early cleavage stages and possibly during oogenesis.     

Modification of histones immediately following synthesis

The acetylation of newly synthesized histones has been studied by exploiting the ability of sodium butyrate to inhibit deacetylation. Upon arrival in the nucleus a significant fraction of histones H2a, H2b, and H3 is in the parental unmodified form. In contrast histone H4 first appears primarily in the diacetylated form. During an ensuing 135 min the diacetylated H4 is slowly further modified.     

G1 and S phase mammalian cells synthesize histones at equivalent rates

To determine the effect of cell cycle position on protein synthesis, synchronized cell populations were metabolically labeled and the synthesis of the basic proteins, including histones, was examined by two-dimensional gel electrophoresis. Exponentially growing S49 mouse lymphoma or Chinese hamster ovary (CHO) cells were separated into G1 and S phase populations by centrifugal elutriation, selective mitotic detachment, fluorescence-activated cell sorting, or a combination of these, and pulse-labeled with radiolabeled amino acids. The histone proteins, both free and chromatin-bound, were completely resolved from some 300 other basic polypeptides in whole-cell lysates by a modification of the NEPHGE technique of O'Farrell, Goodman and O'Farrell (1977). Comparisons of matched autoradiograms from samples of G1 and S phase labeled cells revealed an equivalent rate of histone synthesis through the cell cycle of both S49 and CHO cells. Nuclei isolated from G1 phase S49 cells that were pulse-labeled contained between 13 and 15% of the newly synthesized nucleosomal histones present in S phase nuclei. Nuclei prepared from G1 phase cells that were pulse-labeled and then chased for 5 hr contained more than 90% of the labeled nucleosomal histones present in wholecell lysates. It therefore seems likely that differential alterations in the rate of histone synthesis do not occur to a significant degree as cells proceed through the cycle, but the association of newly synthesized histones with DNA takes place after the onset of DNA replication.     

Polypeptide composition of rat high density lipoprotein: characterization by SDS-gel electrophoresis

The histones synthesized by three different interspecies hybrid echinoid embryos have been examined. In all three crosses, the species-specific F1 histone of the paternal parent is found in the chromatin of the hybrid. These findings provide direct evidence for the involvement of newly transcribed mRNA in the synthesis of this protein. F1 histone is a unique protein in that it is the only paternal protein detected in these hybrids. The possibility that the synthesis of histones is controlled differently from that of other proteins is discussed.     

Antimicrobial proteins in chicken reproductive system

Antimicrobial activity was detected in the ovary and oviduct tissues of healthy mature White Leghorn hens, Gallus gallus. Two antimicrobial proteins were purified to homogeneity using acid extraction followed by multiple steps of chromatography and the pure proteins were further characterized biochemically. Peptide mixtures obtained after enzymatic digestion of the chicken antimicrobial proteins were analyzed using peptide mass fingerprinting and partial sequencing by tandem nanoelectrospray mass spectrometry and the proteins were identified as histones H1 and H2B. Chicken histone antimicrobial proteins were active against both Gram-positive and Gram-negative bacteria. The abundance of these proteins in the reproductive tissues and their broad-spectrum antimicrobial nature may indicate their defensive role against pathogens during the follicle development in the ovary and egg formation in the oviduct. The discovery of antimicrobial histones in chicken reproductive system provides further evidence that histones may play a role in innate immunity against microorganisms in a wide range of animal species.     

Analysis of histone deposition on specific DNA elements in living mammalian cells

DNA replication and repair involve the deposition of newly synthesized histones. After their deposition on a specific locus, histones are extensively modified on their protruding N-terminal tails to restore the histone code corresponding to this locus. These processes require a complex machinery of histone chaperones and histone-modifying enzymes. The precise kinetics of these events and the identity of the factors involved are still not clear. Here we present a technique based on transient expression of tagged histones followed by chromatin immunoprecipitation that allows the detection of newly synthesized histone deposition on specific DNA elements in mammalian cells at high resolution. This strategy could have important applications in chromatin dynamic studies.     

Antimicrobial activity of histones from hemocytes of the Pacific white shrimp.

The role of vertebrate histone proteins or histone derived peptides as innate immune effectors has only recently been appreciated. In this study, high levels of core histone proteins H2A, H2B, H3 and H4 were found in hemocytes from the Pacific white shrimp, Litopenaeus vannamei. The proteins were identified by in-gel digestion, mass spectrometry analysis, and homology searching. The L. vannamei histone proteins were found to be highly homologous to histones of other species. Based on this homology, histone H2A was cloned and its N-terminus was found to resemble the known antimicrobial histone peptides buforin I, parasin, and hipposin. Consequently, a 38 amino acid synthetic peptide identical to the N-terminus of shrimp H2A was synthesized and assayed, along with endogenous histones H2A, H2B, and H4, for growth inhibition against Micrococcus luteus. Histone H2A, purified to homogeneity, completely inhibited growth of the Gram-positive bacterium at 4.5 microm while a mixture of histones H2B and H4 was active at 3 microm. In addition, a fraction containing a fragment of histone H1 was also found to be active. The synthetic peptide similar to buforin was active at submicromolar concentrations. These data indicate, for the first time, that shrimp hemocyte histone proteins possess antimicrobial activity and represent a defense mechanism previously unreported in an invertebrate. Histones may be a component of innate immunity more widely conserved, and of earlier origin, than previously thought.     

Purification and properties of the Xenopus Hat1 acetyltransferase: association with the 14-3-3 proteins in the oocyte nucleus.

We have purified the Xenopus histone acetyltransferase Hat1 holoenzyme from oocytes. The holoenzyme contains the catalytic subunit Hat1, the retinoblastoma associated protein RbAp48, and members of the phosphoserine binding family of 14-3-3 proteins. We have determined that the Hat1 holoenzyme specifically acetylates free histone H4 but not nucleosomal histones. RbAp48 is a phosphoprotein that contains a consensus recognition motif for the 14-3-3 proteins. The 14-3-3 proteins provide a regulatory function for the activity of many phosphoproteins. We find that the hugely abundant Hat1 holoenzyme is present in 10 000-fold excess over somatic cell levels. The holoenzyme is localized in the oocyte nucleus where acetylated histones are stored. The oocyte form of the Xenopus Hat1 holoenzyme may represent a specialized storage form of histone acetyltransferase. Following oocyte maturation and subsequent embryogenesis, the Hat1 enzyme is redistributed to the cytoplasm, where new histones are synthesized.     

Temporal association of the herpes simplex virus genome with histone proteins during a lytic infection

Previous work has determined that there are nucleosomes on the herpes simplex virus (HSV) genome during a lytic infection but that they are not arranged in an equally spaced array like in cellular DNA. However, like in cellular DNA, the promoter regions of several viral genes have been shown to be associated with nucleosomes containing modified histone proteins that are generally found associated with actively transcribed genes. Furthermore, it has been found that the association of modified histones with the HSV genome can be detected at the earliest times postinfection (1 h postinfection) and increases up to 3 h postinfection. However from 3 h to 6 h postinfection (the late phase of the replication cycle), the association decreases. In this study we have examined histone association with promoter regions of all kinetic classes of genes. This was done over the time course of an infection in Sy5y cells using sucrose gradient sedimentation, bromodeoxyuridine labeling, chromatin immunoprecipitation assays, Western blot analysis, trypsin and DNase digestion, and quantitative real-time PCR. Because no histones were detected inside HSV type 1 capsids, the viral genome probably starts to associate with histones after being transported from infecting virions into the host nucleus. Promoter regions of all gene classes (immediate early, early, and late) bind with histone proteins at the start of viral gene expression. However, after viral DNA replication initiates, histones appear not to associate with newly synthesized viral genomes.     

Replication-independent histone deposition by the HIR complex and Asf1

The orderly deposition of histones onto DNA is mediated by conserved assembly complexes, including chromatin assembly factor-1 (CAF-1) and the Hir proteins . CAF-1 and the Hir proteins operate in distinct but functionally overlapping histone deposition pathways in vivo . The Hir proteins and CAF-1 share a common partner, the highly conserved histone H3/H4 binding protein Asf1, which binds the middle subunit of CAF-1 as well as to Hir proteins . Asf1 binds to newly synthesized histones H3/H4 , and this complex stimulates histone deposition by CAF-1 . In yeast, Asf1 is required for the contribution of the Hir proteins to gene silencing . Here, we demonstrate that Hir1, Hir2, Hir3, and Hpc2 comprise the HIR complex, which copurifies with the histone deposition protein Asf1. Together, the HIR complex and Asf1 deposit histones onto DNA in a replication-independent manner. Histone deposition by the HIR complex and Asf1 is impaired by a mutation in Asf1 that inhibits HIR binding. These data indicate that the HIR complex and Asf1 proteins function together as a conserved eukaryotic pathway for histone replacement throughout the cell cycle.     

Cytochemical studies on the protamine-type protein transition in sperm nuclei after fertilization and the early embryonic histones of Urechis caupo.

Cytochemical staining characteristics of nuclear histones during postfertilization maturation division and various early embryonic stages in Urechis have been studied. The transition of protamine-type protein to adult histones in the sperm nucleus is accomplished by 15 min after entrance into the egg cytoplasm. Newly synthesized egg proteins migrate into enlarging male and female pronuclei after this transition, followed by pronuclear DNA synthesis and fusion. The shift from protamine-type protein to adult histones, which occurs in the absence of RNA synthesis during the postfertilization maturation division of the egg, may be one of the processes involved in the normal structural reorganization of chromosomes. Such a reorganization is likely to be a prerequisite for chromosome replication and mitosis. No qualitative differences are detected in the stainability of histones of unfertilized eggs and embryos at the cleavage and later stages of development.     

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.